Compositions and methods for monitoring the phosphorylation of natural binding partners

ABSTRACT

This invention relates to methods and compositions for monitoring the interaction of binding partners as a function of the addition or subtraction of a phosphate group to or from one of the binding partners by a protein kinase or phosphatase.

This application is a continuation-in-part of application Ser. No.09/258,981, filed Feb. 26, 1999.

FIELD OF THE INVENTTON

The invention relates to monitoring of phosphorylation ordephosphorylation of a protein.

BACKGROUND OF THE INVENTION

The post-translational modification of proteins has been known for over40 years and since then has become a ubiquitous feature of proteinstructure. The addition of biochemical groups to translated polypeptideshas wide-ranging effects on protein stability, proteinsecondary/tertiary structure, enzyme activity and in more general termson the regulated homeostasis of cells. Such additions include, but arenot limited to, protein phosphorylation and dephosphorylation.

Phosphorylation is a well-studied example of a post-translationalmodification of proteins. There are many cases in which polypeptidesform higher order tertiary structures with like polypeptides(homo-oligomers) or with unalike polypeptides (hetero-oligomers). In thesimplest scenario, two identical polypeptides associate to form anactive homodimer. An example of this type of association is the naturalassociation of myosin II molecules in the assembly of myosin intofilaments.

The dimerization of myosin II monomers is the initial step in seedingmyosin filaments. The initial dimerization is regulated byphosphorylation, the effect of which is to induce a conformationalchange in myosin II secondary structure resulting in the folded 10Smonomer subunit extending to a 6S molecule. This active molecule is ableto dimerize and subsequently to form filaments. The involvement ofphosphorylation of myosin II in this priming event is somewhatcontroversial. Although in higher eukaryotes the conformational changeis dependant on phosphorylation, in Ancanthoamoeba, a lower eukaryote,the post-translational addition of phosphate is not required to effectthe initial dimerization step. It is of note that the dimerizationdomains in myosin II of higher eukaryotes contain the sites forphosphorylation and it is probable that phosphorylation in this regionis responsible for enabling myosin II to dimerize and subsequently formfilaments. In Dictyostelium this situation is reversed in that thephosphorylation sites are outside the dimerization domain andphosphorylation at these sites is required to effect the disassembly ofmyosin filaments. In contrast to both these examples, Acanthoamoebamyosin II is phosphorylated in the dimerization domain but thismodification is not necessary to enable myosin II monomers to dimerizein this species.

By far the most frequent example of post-translational modification isthe addition of phosphate to polypeptides by specific enzymes known asprotein kinases. These enzymes have been identified as importantregulators of the state of phosphorylation of target proteins and havebeen implicated as major players in regulating cellular physiology. Forexample, the cell-division-cycle of the eukaryotic cell is primarilyregulated by the state of phosphorylation of specific proteins, thefunctional state of which is determined by whether or not the protein isphosphorylated. This is determined by the relative activity of proteinkinases which add phosphate and protein phosphatases which remove thephosphate moiety from these proteins. Clearly dysfunction of either thekinases or phosphatases may lead to a diseased state. This is bestexemplified by the uncontrolled cellular division shown by tumor cells.The regulatory pathway is composed of a large number of genes thatinteract in vivo to regulate the phosphorylation cascade that ultimatelydetermines if a cell is to divide or arrest cell division.

Currently there are several approaches to analysing the state ofmodification of target proteins in vivo:

1. In vivo incorporation of labeled (for example, radiolabeled)phosphate, which is added to target proteins. According to one commonprocedure, intracellular ATP pools are labeled with ³²PO₄, which issubsequently incorporated into protein. Analysis of modified proteins istypically performed by electrophoresis and autoradiography, withspecificity enhanced by immunoprecipitation of proteins of interestprior to electrophoresis.

2. Back-labeling. The incorporation of a labeled phosphate (e.g., ³²P)into a protein in vitro to estimate the state of modification in vivo.

3. The use of cell-membrane-permeable protein kinase inhibitors (e.g.,Wortmannin, staurosporine) to block phosphorylation of target proteins.

4. Western blotting, of either 1- or 2-dimensional gels bearing testprotein samples, in which phosphorylation is detected using antibodiesspecific for phosphorylated forms of target proteins.

5. The exploitation of eukaryotic microbial systems to identifymutations in protein kinases and/or protein phosphatases.

These strategies have certain limitations. Monitoring states ofphosphorylation by pulse or steady-state labeling is merely adescriptive strategy to show which proteins are phosphorylated whensamples are separated by gel electrophoresis and visualized byautoradiography. This is unsatisfactory, due to the inability toidentify many of the proteins that are phosphorylated. A degree ofspecificity is afforded to this technique if it is combined withimmunoprecipitation; however, this is of course limited by theavailability of antibodies to target proteins. Moreover, onlyhighly-expressed proteins are readily detectable using this technique,which may fail to identify many low-abundance proteins, which arepotentially important regulators of cellular functions.

The use of kinase inhibitors to block activity is also problematic. Forexample, very few kinase inhibitors have adequate specificity to allowfor the unequivocal correlation of a given kinase with a specific kinasereaction. Indeed, many inhibitors have a broad inhibitory range. Forexample, staurosporine is a potent inhibitor of phospholipid/Ca⁺²dependant kinases. Wortmannin is some what more specific, being limitedto the phosphatidylinositol-3 kinase family. This is clearlyunsatisfactory because more than one biochemical pathway may be affectedduring treatment making the assignment of the effects almost impossible.

Monoclonal antibodies directed against phosphorylated epitopes, exceptin specific cases, exhibit a limitation of specificity comparable tothat observed when in vivo labeling is undertaken. Immunological methodscan only detect phosphorylated proteins globally (e.g., ananti-phosphotyrosine antibody will detect all tyrosine-phosphorylatedproteins) and can only describe a steady state, rather provide areal-time assessment of protein:protein interactions. Such assays alsorequire considerable manpower for processing.

Finally, yeast (Saccharomyces cervisiae and Schizosaccharomyces pombe)has been exploited as a model organism for the identification of genefunction using recessive mutations. It is through research on theeffects of these mutations that the functional specificities of manyprotein kinases have been elucidated. However, these molecular genetictechniques are not easily transferable to higher eukaryotes, which arediploid and therefore not as genetically tractable as these lowereukaryotes.

Recent research into the sites of protein phosphorylation has revealed anumber of sequence specific motifs which, when phosphorylated ordephosphorylated, promote interaction with selected target proteins toeither induce or inhibit activity of either the phosphorylatedpolypeptide or the target polypeptide.

For example, and not by way of limitation, many proteins involved inintracellular signal transduction have been shown to contain a domaincomprising a sequence of approximately 100 amino acids; this sequence istermed the Src homology two (SH2) domain. SH2 domains bind targetpolypeptides that contain phosphorylated tyrosine. This binding isdependent on the primary amino acid sequence around the phosphotyrosinein the target protein and several peptide sequences which, whenphosphorylated, bind to an SH2 domain have been identified (see e.g.,Songyang et al., 1993, Cell, 72: 767-778). Non-limiting examples of suchsequences include FLPVPEYINQSV, SEQ ID NO: 1, a sequence found in humanECF receptor, and AVGNPEYLNTVQ, SEQ ID NO:2, a sequence found in humanEGF receptor, both of which are autophosphorylated growth factorreceptors which stimulate the biochemical signaling pathways thatcontrol gene expression, cytoskeletal architecture and cell metabolism.Both of these sequences interact with SH2 domains found in the Sen-5adapter protein.

The tumor suppressor protein p53, becomes activated by a transcriptionfactor in response to DNA damage. A DNA-dependent protein kinase(DNA-PK) that is activated in response to breaks in DNA is thought to beregulator of p53 activity (Woo et al., 1998, Nature, 394: 700-704). Thedata described by Woo et al. indicate that the phosphorylation of p53 byDNA-PK serves a dual purpose insofar as phosphorylation promotes thebinding of p53 to DNA and also prevents p53 inactivation by MDM2. Ap53-derived peptide sequence EPPLSQEAFADLWKK, SEQ ID NO:3 is identifiedas the site of phosphorylation in p53 that (when phosphorylated)prevents the interaction of p53 with MIDM2.

An example of heterodimer association is described in patent applicationnumber WO92/00388. It describes an adenosine 3:5 cyclic monophosphate(cAMP) dependent protein kinase which is a four-subunit enzyme beingcomposed of two catalytic polypeptides (C) and two regulatorypolypeptides (R). In nature the polypeptides associate in astoichiometry of R₂C₂. In the absence of cAMP the R and C subunitsassociate and the enzyme complex is inactive. In the presence of cAMPthe R subunit functions as a ligand for cAMP resulting in dissociationof the complex and the release of active protein kinase. The inventiondescribed in WO92/00388 exploits this association by addingfluorochromes to the R and C subunits.

The polypeptides are labeled (or ‘tagged’) with fluorophores havingdifferent excitation/emission wavelengths. The excitation and emissionof one such fluorophore effects a second excitation/emission event inthe second fluorophore. By monitoring the fluorescence emission of eachfluorophore, which reflects the presence or absence of fluorescenceenergy transfer between the two, it is possible to derive theconcentration of cAMP as a function of the level of association betweenthe R and C. Therefore, the natural affinity of the C subunit for the Rsubunit has been exploited to monitor the concentration of a specificmetabolite, namely cAMP.

The prior art teaches that intact, fluorophore-labeled proteins canfunction as reporter molecules for monitoring the formation ofmulti-subunit complexes from protein monomers; however, in each case,the technique relies on the natural ability of the protein monomers toassociate.

Tsien et al. (WO97/28261) teach that fluorescent proteins having theproper emission and excitation spectra that are brought into physicallyclose proximity with one another can exhibit fluorescence resonanceenergy transfer (“FRET”). The invention of WO97/28261 takes advantage ofthat discovery to provide tandem fluorescent protein constructs in whichtwo fluorescent protein labels capable of exhibiting FRET are coupledthrough a linker to form a tandem construct. In the assays of Tsien etal., protease activity is monitored using FRET to determine the distancebetween fluorophores controlled by a peptide linker and subsequenthydrolysis thereof. Other applications rely on a change in the intrinsicfluorescence of the protein as in the kinase assays of WO98/06737.

The present invention instead encompasses monitoring of the associationof polypeptides, as described herein, which are labeled with fluorescent(protein and chemical) or other labels. FRET, a non-limiting example ofa detection method of use in the invention, indicates the proximity oftwo labeled polypeptide binding partners, which labeled partnersassociate either in the presence or absence of post-translationaladdition/removal of a phosphate group to/from a natural binding domainpresent in at least one of the partners, but not into the fluorophore,reflecting the phosphorylation state of one or both of the bindingpartners and, consequently, the level of activity of a protein kinase orphosphatase.

There is a need in the art for efficient means of monitoring and/ormodulating post-translational protein phosphorylation and/ordephosphorylation. Further, there is a need to develop a techniquewhereby the addition/removal of a phosphate group can be monitoredcontinuously during real time to provide a dynamic assay system thatalso has the ability to resolve spatial information.

SUMMARY OF THE INVENTION

The invention provides natural binding domains, sequences andpolypeptides, as well as kits comprising these molecules and assays ofenzymatic function in which they are employed as reporter molecules. Asused herein in reference to a polypeptide component of assays of theinvention, the term “natural” refers both to the existence of such anamino acid sequence, whether contiguous or non-contiguous, in nature aswell as to the phosphorylation-dependent binding of that component to asecond polypeptide or binding partner, and does not relate to attributesof such a polypeptide other than such binding.

One aspect of the invention is an isolated natural binding domain and abinding partner therefor, wherein the isolated natural binding domainincludes a site for post-translational phosphorylation and binds thebinding partner in a manner dependent upon phosphorylation ordephosphorylation of the site.

The invention also provides a method for monitoring activity of anenzyme comprising performing a detection step to detect binding of anisolated natural binding domain and a binding partner therefor as aresult of contacting one or both of the isolated natural binding domainand the binding partner with the enzyme, wherein the isolated naturalbinding domain includes a site for post-translational phosphorylationand binds the binding partner in a manner dependent upon phosphorylationof the site and wherein detection of binding of the isolated naturalbinding domain and the binding partner as a result of the contacting isindicative of enzyme activity.

An enzyme to be assayed according to the invention is a protein kinaseor a phosphatase.

The invention additionally encompasses a method for monitoring activityof an enzyme comprising performing a detection step to detectdissociation of an isolated natural binding domain from a bindingpartner therefor as a result of contacting one or both of the isolatednatural binding domain and the binding partner with the enzyme, whereinthe isolated natural binding domain includes a site forpost-translational phosphorylation and binds the binding partner in amanner dependent upon phosphorylation of the site and wherein detectionof dissociation of the isolated natural binding domain from the bindingpartner as a result of the contacting is indicative of enzyme activity.

As used herein, the term “binding domain” in a three-dimensional senserefers to the amino acid residues of a first polypeptide required forphosphorylation-dependent binding between the first polypeptide and itsbinding partner. The amino acids of a “binding domain” may be eithercontiguous or non-contiguous and may form a binding pocket forphosphorylation-dependent binding. A domain must include at least 1amino acid, but may include 2 or more amino acids, preferably at least 4amino acids, which are contiguous or non-contiguous, but are necessaryfor phosphorylation-dependent binding to the binding partner. A bindingdomain will not include a natural full-length polypeptide, but willinclude a subset of the amino acids of a full-length polypeptide,wherein the subset may include a number of amino acids as high as onefewer than the length of a given natural full-length polypeptide.

A binding domain which is of use in the invention is a “natural bindingdomain” (i.e., a binding domain that exhibits phosphorylation-dependentbinding to a binding partner in nature). A natural binding domain of usein the invention may be isolated or may be present in the context of alarger polypeptide molecule (i.e., one which comprises amino acids otherthan those of the natural binding domain), which molecule may be eithernaturally-occurring or recombinant and, in the case of the latter, maycomprise either natural or non-natural amino acid sequences outside thebinding domain.

As used herein with regard to phosphorylation or dephosphorylation of apolypeptide, the term “site” refers to an amino acid or amino acidsequence of a natural binding domain or a binding partner which isrecognized by (i.e., a signal for) a kinase or phosphatase for thepurpose of phosphorylation or dephosphorylation (i.e., addition orremoval of a phosphate moiety) of the polypeptide or a portion thereof.A “site” additionally refers to the single amino acid which isphosphorylated or dephosphorylated. It is contemplated that a sitecomprises a small number of amino acids, as few as one but typicallyfrom 2 to 10, less often up to 30 amino acids, and further that a sitecomprises fewer than the total number of amino acids present in thepolypeptide.

In an enzymatic assay of the invention, a “site”, for post-translationalphosphorylation or dephosphorylation may be present on either or both ofthe isolated natural binding domain or the binding partner therefor. Ifsuch sites are present on both the isolated natural binding domain andits binding partner, binding between the natural binding domain and thebinding partner, or between two natural binding domains, may bedependent upon the phosphorylation or dephosphorylation state of eitherone or both sites. If a single polypeptide chain comprises the naturalbinding domain and the binding partner (or two natural binding domains),the state of phosphorylation or dephosphorylation of one or both siteswill determine whether binding occurs.

A site suitable for addition or removal of a phosphate moiety is presentwithin an isolated natural binding domain or binding partner thereof ofthe invention at a position such that formation of a complex between theisolated natural binding domain and its binding partner is dependentupon the presence or absence of the phosphate moiety; and preferablydoes not overlap with an amino acid which is part of a fluorescent tagor other detectable label (including, but not limited to, a radioactivelabel) or quencher.

Similarly, the amino acid that includes a phosphate moiety may bepositioned anywhere within the isolated natural binding domain such thatbinding of the isolated natural binding domain and its binding partneris dependent upon the presence or absence of the phosphate moiety.

As used herein, “phosphorylation” and “dephosphorylation” refer to theaddition or removal of a phosphate moiety to/from a polypeptide,respectively. As used herein, the term “post-translational modification”refers to the addition or removal of a phosphate moiety and does notrefer to other post-translational events which do not involve additionor removal of a phosphate moiety, and thus does not include simplecleavage of the reporter molecule polypeptide backbone by hydrolysis ofa peptide bond.

As used herein, the term “moiety” refers to a post-translationally addedor removed phosphate (PO₄) group; the terms “moiety” and “group” areused interchangeably.

As used herein, the term “binding partner” refers to a polypeptide orfragment thereof (a peptide) that binds to a binding domain, sequence orpolypeptide, as defined herein, in a manner which is dependent upon thestate of phosphorylation of a site for phosphorylation ordephosphorylation which is, at a minimum, present upon the bindingdomain, sequence or polypeptide; the binding partner itself may,optionally, comprise such a site and binding between the binding domain,fragment or polypeptide with its corresponding binding partner may,optionally, depend upon modification of that site. A binding partnerdoes not necessarily have to contain a site for phosphorylation ordephosphorylation if such an site is not required to be present on itfor modification-dependent association between it and a binding domain,sequence or polypeptide. Binding partners of use in the invention arethose which are found in nature and exhibit naturalphosphorylation-dependent binding to a natural binding domain, sequenceor polypeptide of the invention as defined herein. In one embodiment ofthe invention, a binding partner is shorter (i.e., by at least oneN-terminal or C-terminal amino acid) than the natural full-lengthpolypeptide.

As used herein, the term “associates” or “binds” refers to a naturalbinding domain as described herein and its binding partner, having abinding constant sufficiently strong to allow detection of binding byFRET or other detection means, which are in physical contact with eachother and have a dissociation constant (Kd) of about 10 μM or lower. Thecontact region may include all or parts of the two molecules. Therefore,the terms “substantially dissociated” and “dissociated” or“substantially unbound” or “unbound” refer to the absence or loss ofcontact between such regions, such that the binding constant is reducedby an amount which produces a discernable change in a signal compared tothe bound state, including a total absence or loss of contact, such thatthe proteins are completely separated, as well as a partial absence orloss of contact, so that the body of the proteins are no longer in closeproximity to each other but may still be tethered together or otherwiseloosely attached, and thus have a dissociation constant greater than 10μM (Kd). In many cases, the Kd will be in the mM range. The terms“complex”, “dimer”, “multimer” and “oligomer” as used herein, refer tothe natural binding domain and its binding partner in the associated orbound state. More than one molecule of each of the two or more proteinsmay be present in a complex, dimer, multimer or oligomer according tothe methods of the invention.

As used herein in reference to a natural binding domain or otherpolypeptide, the term “isolated” refers to a molecule or population ofmolecules that is substantially pure (i.e., free of contaminatingmolecules of unlike amino acid sequence).

As used herein in reference to the purity of a molecule or populationthereof, the term “substantially” refers to that which is at least 50%,preferably 60-75%, more Jo preferably from 80-95% and, most preferably,from 98-100% pure.

“Naturally-occurring” as used herein, as applied to a polypeptide orpolynucleotide, refers to the fact that the polypeptide orpolynucleotide can be found in nature. One such example is a polypeptideor polynucleotide sequence that is present in an organism (including avirus) that can be isolated form a source in nature.

The term “synthetic”, as used herein, is defined as any amino- ornucleic acid sequence which is produced via chemical synthesis.

In an assay of the invention, post-translational phosphorylation isreversible, such that repeating cycles of addition and removal of aphosphate moiety may be observed, although such cycles may not occur ina living cell found in nature.

An advantage of assays of the invention is that they may, if desired, beperformed in “real time”. As used herein in reference to monitoring,measurements or observations in assays of the invention, the term “realtime” refers to that which is performed contemporaneously with themonitored, measured or observed events and which yields a result of themonitoring, measurement or observation to one who performs itsimultaneously, or effectively so, with the occurrence of a monitored,measured or observed event. Thus, a “real time” assay or measurementcontains not only the measured and quantitated result, such asfluorescence, but expresses this in real time, that is, in hours,minutes, seconds, milliseconds, nanoseconds, picoseconds, etc. Shortertimes exceed the instrumentation capability; further, resolution is alsolimited by the folding and binding kinetics of polypeptides.

As used herein, the term “binding sequence” refers to that portion of apolypeptide comprising at least 1, preferably at least 2, morepreferably at least 4, and up to 8, 10, 100 or even 1000 contiguous(i.e., covalently linked by peptide bonds) amino acid residues, that aresufficient for phosphorylation-dependent binding to a binding partner. Abinding sequence will not include a natural full-length polypeptide, butwill include a subset of the amino acids of a full-length polypeptide,wherein the subset may include a number of amino acids as high as onefewer than the length of a given natural full-length polypeptide.

As used herein in reference to those binding sequences that are of usein the invention, the term “natural binding sequence” refers to abinding sequence, as defined above, which consists of an amino acidsequence which is found in nature and which is naturally dependent uponthe phosphorylation state of a site for post-translationalphosphorylation found within it for binding to a binding partner. A“natural binding sequence” may be present either in isolation or in thecontext of a larger polypeptide molecule, which molecule may benaturally-occurring or recombinant. If present, amino acids outside ofthe binding sequence may be either natural, i.e., from the samepolypeptide sequence from which the fragment is derived, or non-natural,i.e., from another (different) polypeptide or from a sequence that isnot derived from any known polypeptide. In assays of the invention, abinding sequence and its binding partner may exist either on twodifferent polypeptide chains or on a single polypeptide chain.

As used herein, the term “binding polypeptide” refers to a moleculecomprising multiple binding sequences, as defined above. A bindingpolypeptide of use in the invention is a “natural binding polypeptide”,in which the component binding sequences are natural binding sequences,as defined above (e.g., wherein the binding sequences are derived from asingle, naturally-occurring polypeptide molecule), and are bothnecessary and, in combination, sufficient to permit phosphorylationstate-dependent binding of the binding polypeptide to its bindingpartner, wherein the sequences of the binding polypeptide are eithercontiguous or are non-contiguous. As used herein in reference to thecomponent binding sequences of a binding polypeptide, the term“non-contiguous” refers to binding sequences which are linked byintervening naturally-occurring, as defined herein, or non-natural aminoacid sequences or other chemical or biological linker molecules such areknown in the art. The amino acids of a polypeptide that do notsignificantly contribute to the natural phosphorylation-state-dependentbinding of that polypeptide to its binding partner may be those aminoacids which are naturally present and link the binding sequences in abinding polypeptide or they may be derived from a different naturalpolypeptide or may be wholly unknown in nature. In assays of theinvention, a binding polypeptide and its binding partner (which may,itself, be a binding domain, sequence or polypeptide, as defined herein)may exist on two different polypeptide chains or on a single polypeptidechain. According to the invention, a natural binding polypeptide, like apolypeptide as defined above, is not a full-length natural polypeptidechain, but instead comprises a subset that encompasses up to one fewerthan the total number of amino acids in a natural polypeptide chain.

As used herein, the terms “polypeptide” and “peptide” refer to a polymerin which the monomers are amino acids and are joined together throughpeptide or disulfide bonds. The terms subunit and domain also may referto polypeptides and peptides having biological function. A peptideuseful in the invention will at least have a binding capability, i.e,with respect to binding as- or to a binding partner, and also may haveanother biological function that is a biological function of a proteinor domain from which the peptide sequence is derived. “Polypeptide”refers to a naturally-occurring amino acid chain comprising a subset ofthe amino acids of a full-length protein, wherein the subset comprisesat least one fewer amino acid than does the full-length protein, or a“fragment thereof” or “peptide”, such as a selected region of thepolypeptide that is of interest in a binding assay and for which abinding partner is known or determinable. “Fragment thereof” thus refersto an amino acid sequence that is a portion of a full-lengthpolypeptide, between about 8 and about 1000 amino acids in length,preferably about 8 to about 300, more preferably about 8 to about 200amino acids, and even more preferably about 10 to about 50 or 100 aminoacids in length. “Peptide” refers to a short amino acid sequence that is10-40 amino acids long, preferably 10-35 amino acids. Additionally,unnatural amino acids, for example, β-alanine, phenyl glycine andhomoarginine may be included. Commonly-encountered amino acids which arenot gene-encoded may also be used in the present invention. All of theamino acids used in the present invention may be either the D- or L-optical isomer. The L-isomers are preferred. In addition, otherpeptidomimetics are also useful, e.g. in linker sequences ofpolypeptides of the present invention (see Spatola, 1983, in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed.,Marcel Dekker, New York, p. 267).

As used herein, the terms “protein”, “subunit” and “domain” refer to alinear sequence of amino acids which exhibits biological function. Thislinear sequence does not include full-length amino acid sequences (e.g.those encoded by a full-length gene or polynucleotide), but does includea portion or fragment thereof, provided the biological function ismaintained by that portion or fragment. The terms “subunit” and “domain”also may refer to polypeptides and peptides having biological function.A peptide useful in the invention will at least have a bindingcapability, i.e, with respect to binding as or to a binding partner, andalso may have another biological function that is a biological functionof a protein or domain from which the peptide sequence is derived.“Polynucleotide” refers to a polymeric form of nucleotides of at least10 bases in length and up to 1,000 bases or even more, eitherribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. The term includes single and double stranded formsof DNA.

Preferably, with regard to the natural binding domain and/or bindingpartner therefor, phosphorylation or dephosphorylation is performed byan enzyme which is a kinase or a phosphatase, respectively.

It is preferred that phosphorylation of the site prevents binding of theisolated natural binding domain to the binding partner.

As used herein, the term “prevents” refers to a reduction of at least10%, preferably 20-40%, more preferably 50-75% and, most preferably,80-100% of binding of the isolated natural binding domain to the bindingpartner therefor.

Preferably, phosphorylation of the site promotes binding of the isolatednatural binding domain to the binding partner.

As used herein with regard to protein:protein binding, the term“promotes” refers to that which causes an increase in binding of thenatural binding domain and its binding partner of at least two-fold,preferably 10- to 20-fold, highly preferably 50- to 100-fold, morepreferably from 200- to 1000-fold, and, most preferably, from 200 to10,000-fold.

It is preferred that dephosphorylation of the site prevents binding ofthe isolated natural binding domain to the binding partner.

It is additionally preferred that dephosphorylation of the site promotesbinding of the isolated natural binding domain to the binding partner.

In a preferred embodiment, at least one of the isolated natural bindingdomain and the binding partner comprises a detectable label.

Preferably, the detectable label emits light.

More preferably, the light is fluorescent.

It is preferred that one of the isolated natural binding domain and thebinding partner therefor comprises a quencher for the detectable label.Labels of use in the invention include, but are not limited to, aradioactive label, a fluorescent label and a quencher for either.

A “fluorescent label”, “fluorescent tag” or “fluorescent group” refersto either a fluorophore or a fluorescent protein or fluorescent fragmentthereof. “Fluorescent protein” refers to any protein which fluoresceswhen excited with appropriate electromagnetic radiation. This includes aprotein whose amino acid sequence is either natural or engineered. A“fluorescent protein” is a full-length fluorescent protein orfluorescent fragment thereof. By the same token, the term “linker”refers to that which is coupled to both the donor and acceptor proteinmolecules, such as an amino acid sequence joining two natural bindingdomains or a disulfide bond between two polypeptides.

It is contemplated that with regard to fluorescent labels employed inFRET, the reporter labels are chosen such that the emission wavelengthspectrum of one (the “donor”) is within the excitation wavelengthspectrum of the other (the “acceptor”). With regard to a fluorescentlabel and a quencher employed in a single-label detection procedure inan assay of the invention, it is additionally contemplated that thefluorophore and quencher are chosen such that the emission wavelengthspectrum of the fluorophore is within the absorption spectrum of thequencher, such that when the fluorophore and the quencher with which itis employed are brought into close proximity by binding of the naturalbinding domain, sequence or polypeptide upon which one is present withthe binding partner comprising the other, detection of the fluorescentsignal emitted by the fluorophore is reduced by at least 10%, preferably20-50%, more preferably 70-90% and, most preferably, by 95-100%. Atypical quencher reduces detection of a fluorescent signal byapproximately 80%.

Another aspect of the invention is a kit comprising an isolated naturalbinding domain and a binding partner therefor, wherein the isolatednatural binding domain includes a site for post-translationalphosphorylation and binds the binding partner in a manner dependent uponphosphorylation of the site, and packaging material therefor.

It is preferred that the kit further comprises a buffer which permitsphosphorylation-dependent binding of the isolated natural binding domainand the binding partner.

As used herein, the term “buffer” refers to a medium which permitsactivity of the protein kinase or phosphatase used in an assay of theinvention, and is typically a low-ionic-strength buffer or otherbiocompatible solution (e.g., water, containing one or more ofphysiological salt, such as simple saline, and/or a weak buffer, such asTris or phosphate, or others as described hereinbelow), a cell culturemedium, of which many are known in the art, or a whole or fractionatedcell lysate. Such a buffer permits phosphorylation-dependent binding ofa natural binding domain of the invention and a binding partner thereforand, preferably, inhibits degradation and maintains biological activityof the reaction components. Inhibitors of degradation, such as proteaseinhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease inhibitors(e.g., DEPC) are well known in the art. Lastly, an appropriate buffermay comprise a stabilizing substance such as glycerol, sucrose orpolyethylene glycol.

As used herein, the term “physiological buffer” refers to a liquidmedium that mimics the salt balance and pH of the cytoplasm of a cell orof the extracellular milieu, such that post-translational proteinmodification reactions and protein:protein binding are permitted tooccur in the buffer as they would in vivo.

Preferably, the buffer permits phosphorylation or dephosphorylation ofthe site by a kinase or a phosphatase, respectively.

In a preferred embodiment, the kit further comprises one or both of akinase and a phosphatase.

It is preferred that the kit further comprises a substrate for thephosphatase or kinase, the substrate being MgATP.

It is contemplated that at least a part of a substrate of an enzyme ofuse in an assay of the invention is transferred to a phosphorylationsite on an isolated polypeptide of the invention. As used herein, theterm “at least a part of a substrate” refers to a portion (e.g., amoiety or a group, as defined above) which comprises less than the wholeof the substrate for the enzyme, the transfer of which portion to aphosphorylation site on an isolated polypeptide, both as defined above,is catalyzed by the enzyme.

It is additionally preferred that the kit further comprises a cofactorfor one or both of the kinase or phosphatase. Cofactors of use in theinvention include, but are not limited to, cAMP, phosphotidylserine,diolein, Mn²⁺ and Mg²⁺.

Preferably, at least one of the isolated natural binding domain and thebinding partnercomprises a detectable label.

It is preferred that the detectable label emits light, and morepreferred that the light is fluorescent.

An enzyme (e.g., a protein kinase or phosphatase) of use in theinvention may be natural or recombinant or, alternatively, may bechemically synthesized. If either natural or recombinant, it may besubstantially pure (i.e., present in a population of molecules in whichit is at least 50% homogeneous), partially purified (i.e., representedby at least 1% of the molecules present in a fraction of a cellularlysate) or may be present in a crude biological sample.

As used herein, the term “sample” refers to a collection of inorganic,organic or biochemical molecules which is either found in nature (e.g.,in a biological- or other specimen) or in an artificially-constructedgrouping, such as agents which might be found and/or mixed in alaboratory. Such a sample may be either heterogeneous or homogeneous.

As used herein, the interchangeable terms “biological specimen” and“biological sample” refer to a whole organism or a subset of itstissues, cells or component parts (e.g. body fluids, including but notlimited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluidand semen). “Biological sample” further refers to a homogenate, lysateor extract prepared from a whole organism or a subset of its tissues,cells or component parts, or a fraction or portion thereof. Lastly,“biological sample” refers to a medium, such as a nutrient broth or gelin which an organism has been propagated, which contains cellularcomponents, such as proteins or nucleic acid molecules.

As used herein, the term “organism” refers to all cellular life-forms,such as prokaryotes and eukaryotes, as well as non-cellular, nucleicacid-containing entities, such as bacteriophage and viruses.

In a method as described above, it is preferred that at least one of theisolated natural binding domain and the binding partner is labeled witha detectable label.

Preferably, the label emits light and, more preferably, the light isfluorescent.

In another preferred embodiment, the detection step is to detect achange in signal emission by the detectable label.

It is preferred that the method further comprises exciting thedetectable label and monitoring fluorescence emission.

It is additionally preferred that the method further comprises the step,prior to or after the detection step, of contacting the isolated naturalbinding domain and the binding partner with an agent which modulates theactivity of the enzyme.

As used herein with regard to a biological or chemical agent, the term“modulate” refers to enhancing or inhibiting the activity of a proteinkinase or phosphatase in an assay of the invention; such modulation maybe direct (e.g. including, but not limited to, cleavage of- orcompetitive binding of another substance to the enzyme) or indirect(e.g. by blocking the initial production or, if required, activation ofthe kinase or phosphatase). “Modulation” refers to the capacity toeither increase or decease a measurable functional property ofbiological activity or process (e.g., enzyme activity or receptorbinding) by at least 10%, 15%, 20%, 25%, 50%, 100% or more; suchincrease or decrease may be contingent on the occurrence of a specificevent, such as activation of a signal transduction pathway, and/or maybe manifest only in particular cell types.

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues, or even an inorganicelement or molecule. Modulators are evaluated for potential activity asinhibitors or activators (directly or indirectly) of a biologicalprocess or processes (e.g., agonist, partial antagonist, partialagonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitorsof neoplastic transformation or cell proliferation, cellproliferation-promoting agents, and the like) by inclusion in screeningassays described herein. The activities (or activity) of a modulator maybe known, unknown or partially-known. Such modulators can be screenedusing the methods described herein.

The term “candidate modulator” refers to a compound to be tested by oneor more screening method(s) of the invention as a putative modulator.Usually, various predetermined concentrations are used for screeningsuch as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM, as described more fullyhereinbelow. Test compound controls can include the measurement of asignal in the absence of the test compound or comparison to a compoundknown to modulate the target.

The invention additionally provides a method of screening for acandidate modulator of enzymatic activity of a kinase or a phosphatase,the method comprising contacting an isolated natural binding domain, abinding partner therefor and an enzyme with a candidate modulator of thekinase or phosphatase, wherein the natural binding domain includes asite for post-translational phosphorylation and binds the bindingpartner in a manner that is dependent upon phosphorylation ordephosphorylation of the site by the kinase or phosphatase and whereinat least one of the isolated natural binding domain and the bindingpartner comprises a detectable label, and monitoring the binding of theisolated natural binding domain to the binding partner, wherein bindingor dissociation of the isolated natural binding domain and the bindingpartner as a result of the contacting is indicative of modulation ofenzymatic activity by the candidate modulator of the kinase orphosphatase.

Preferably, the detectable label emits light.

More preferably, the light is fluorescent.

It is preferred that the monitoring comprises measuring a change inenergy transfer between a detectable label present on the isolatednatural binding domain and a detectable label present on the bindingpartner.

A final aspect of the invention is a method of screening for a candidatemodulator of enzymatic activity of a kinase or a phosphatase, the methodcomprising contacting an assay system with a candidate modulator ofenzymatic activity of a kinase or phosphatase, and monitoring binding ofan isolated natural binding domain and a binding partner therefor in theassay system, wherein the isolated natural binding domain includes asite for post-translational phosphorylation and binds the bindingpartner in a manner that is dependent upon phosphorylation ordephosphorylation of the site by a kinase or phosphatase in the assaysystem, wherein at least one of the isolated natural binding domain andthe binding partner comprises a detectable label, and wherein binding ordissociation of the isolated natural binding domain and the bindingpartner as a result of the contacting is indicative of modulation ofenzymatic activity by the candidate modulator of a the kinase orphosphatase.

It is highly preferred that in any of the above methods, the methodcomprises real-time observation of association of an isolated naturalbinding domain and its binding partner.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 diagrams double- and single-chain enzymatic assay formats of theinvention.

FIG. 2 presents a schematic overview of FRET in an assay of theinvention.

FIG. 3 presents monomer:excimer fluorescence.

FIG. 4 demonstrates the results of FRET between ZAP70-GFP and arhodamine labelled TCRζ derived peptide.

FIG. 5 demonstrates the dependence of YOP activity on differentconcentrations of TCRζ peptide.

FIG. 6 presents the results of a FRET based assay for measuringinhibition of YOP by sodium orthovanadate.

FIG. 7 demonstrates detection of binding of Chk1 phosphorylated,fluorescein labelled Chktide to 14-3-3ζ by fluorescence polarisation.

FIG. 8 demonstrates inhibition of Chk1 phosphorylation of Chktidepeptide by EDTA.

FIG. 9 presents the results of a real time assay for Chk1 activitymonitoring the fluorescence polarisation of fluorescein labelled Chktidesubstrate binding to 14-3-3ζ protein.

FIG. 10 demonstrates that Chk1 activity measured by Chktide:14-3-3binding is dependent on ATP and the presence of 14-3-3ζ protein.

FIG. 11 demonstrates inhibition of Chk1 phosphorylation of fluoresceinlabelled Chktide by EDTA.

FIG. 12 presents the results of an assay for Chk1 phosphorylation ofChktide peptide as measured by 14-3-3ε binding.

FIG. 13 demonstrates phosphatase λ activity as measured bydephosphorylation of fluorescein labelled Chktide and decreased bindingto 14-3-3ζ.

FIG. 14 demonstrates phosphatase λ activity as measured bydephosphorylation of fluorescein labelled Chktide and decreased bindingto 14-3-3ε.

FIG. 15 presents a time course of Chk1 and PKA activity measured usingfluorescence polarisation.

FIG. 16 demonstrates detection of peptide phosphorylation by Src kinase,by measuring FRET between ZAP-GFP and a rhodamine labelled substratepeptide.

FIG. 17 demonstrates detection of SHPS-1 derived peptide phosphorylationby Src, and binding of SHPS-1 to SHP2-GFP partner.

FIG. 18 demonstrates YOP mediated reversal of FRET between SHP2-GFP andrhodamine labelled, phosphorylated, SHPS-1 peptide.

FIG. 19 presents detection of Src inhibition by staurosporine using aFRET-based assay between rhodamine labelled SHPS-1 and SHP2-GFP.

FIG. 20 presents the results of a real-time, FRET-based assay, measuringSrc phosphorylation of SHPS-1 peptide.

DESCRIPTION

The invention is based upon the discovery that a natural binding domain,sequence or polypeptide, as defined above, associates with a bindingpartner to form a complex or dissociates from a binding partner, in amanner that is dependent upon the presence or absence of a phosphatemoiety, and that is detectable and measurable in a highly sensitivemanner that may be observed in real time.

Polypeptides of use in the invention

The invention provides reporter molecules and assays for measuring theactivity of protein kinases and phosphatases. These reporter moleculesare naturally-occurring polypeptides which include natural bindingdomains, natural binding sequences and natural binding polypeptides,each as defined above, which are used in assays of the invention incombination with polypeptide binding partners, also as defined above.

Minimally, such a reporter molecule comprises or consists of a naturalbinding domain. The amino acids of a natural binding domain are thosewhich are necessary for phosphorylation-dependent binding of themolecule comprising or consisting of the natural binding domain with abinding partner, whether such a partner is present on the same or adifferent polypeptide chain as the natural binding domain. Such aminoacids may include points of direct contact between the domain and thebinding partner, those which are recognized and/or modified (i.e.,phosphorylated or dephosphorylated) by a kinase or phosphatase and thosewhich maintain the three-dimensional structure or charge of the bindingdomain in a manner which permits phosphorylation and/ordephosphorylation and the consequent phosphorylation- and/ordephosphorylation-dependent binding of the domain to the bindingpartner. The amino acids of a natural binding domain may be contiguousor may be separated by non-domain amino acids; such non-domain residuesmay be either those which are naturally present between the amino acidsof the natural binding domain or which are non-natural. In cases inwhich non-natural amino acids are found interspersed with those of anatural binding domain, such non-natural residues will be residues whichdo not substantially (that is, measurably) alter the naturalphosphorylation-dependent binding of the natural binding domain to itsbinding partner.

A second reporter molecule of use in the invention is that whichcomprises or consists of a naturally-occurring stretch of contiguousamino acids sufficient for phosphorylation-dependent binding to abinding partner, as defined above, i.e., at least the minimum number ofcontiguous amino acids required to encompass a natural binding domain.The phosphorylation-dependence of such a molecule, referred to herein asa “natural binding sequence”, is, itself natural. A reporter molecule ofthe invention may either consist of or comprise a natural bindingsequence. In the latter case, amino acids outside of the natural bindingsequence do not substantially influence phosphorylation-dependentbinding of the natural binding domain to the binding partner.

Lastly, a reporter molecule of use in the invention may be a “naturalbinding polypeptide”, as defined above. Such a polypeptide moleculecomprises or consists of multiple natural binding domains (above), whichdomains are, either individually or in concert with one another,sufficient to permit natural, phosphorylation-dependent binding of thenatural binding polypeptide to a binding partner.

By monitoring the association or dissociation of a natural bindingdomain, sequence or polypeptide and its binding partner in the presenceof a known or candidate protein kinase or phosphatase, the activity ofsuch an enzyme can be measured. In such assays, one or both of thenatural binding domain, sequence or polypeptide and its binding partnercomprises a detectable label including, but not exclusively, afluorescent or other light-emitting label, which may be either chemicalor proteinaceous. By measuring changes in signal emission or absorptionbefore and after addition to the mixture comprising the natural bindingdomain, sequence or polypeptide and its binding partner of the enzyme tobe assayed, the extent of phosphorylation can be calculated. Animportant feature of the invention is that such measurements (e.g., of ashift in FRET) can be performed in real-time. This allows for sensitiveassessment of enzyme reaction kinetics based upon the rate of change ofthe protein-binding-dependent signal emission or absorption by thelabel(s).

Assays in which the above reporter molecules are used according to theinvention may be performed either in double- or single-chain format(FIG. 1). In double-chain format, natural binding domain, sequence orpolypeptide is comprised by a different polypeptide chain from thatcomprising or consisting of the binding partner and is not otherwisecovalently linked to it. In single-chain format, the natural bindingdomain, sequence or polypeptide is covalently linked to its bindingpartner, either through an intervening amino acid sequence or a chemicallinker.

The binding partner of a natural binding domain, sequence or polypeptidemay, itself, be a natural binding domain, sequence or polypeptide asdefined herein. If so, binding of the two molecules may depend upon thephosphorylation state of one or both in a manner that is comparable tothat found in nature.

Methods by which assays of the invention are performed are described indetail in the following sections and in the Examples.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g, in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., which is incorporated herein by reference), chemical methods,pharmaceutical formulation and delivery and treatment of patients.

Methods by which to detect protein:protein binding in assays of theinvention

Methods of detecting the phosphorylation-dependent binding of a naturalbinding domain, sequence or polypeptide and a binding partner in anassay of the invention most usefully, although not exclusively, arethose which employ light-emitting labels. Several such techniques aredescribed below.

Fluorescence energy resonance transfer (FRET)

A tool with which to assess the distance between one molecule andanother (whether protein or nucleic acid) or between two positions onthe same molecule is provided by the technique of fluorescence resonanceenergy transfer (FRET), which is now widely known in the art (for areview, see Matyus, 1992, J. Photochem. Photobiol. B: Biol., 12:323-337, which is herein incorporated by reference). FRET is aradiationless process in which energy is transferred from an exciteddonor molecule to an acceptor molecule; the efficiency of this transferis dependent upon the distance between the donor an acceptor molecules,as described below. Since the rate of energy transfer is inverselyproportional to the sixth power of the distance between the donor andacceptor, the energy transfer efficiency is extremely sensitive todistance changes. Energy transfer is said to occur with detectableefficiency in the 1-10 nm distance range, but is typically 4-6 nm forfavorable pairs of donor and acceptor.

Radiationless energy transfer is based on the biophysical properties offluorophores. These principles are reviewed elsewhere (Lakowicz, 1983,Principles of Flourescence Spectroscopy, Plenum Press, New York; Jovinand Jovin, 1989, Cell Structure and Function by Microspectrofluorometry,eds. E. Kohen and J. G. Hirschberg, Academic Press, both of which areincorporated herein by reference). Briefly, a fluorophore absorbs lightenergy at a characteristic wavelength. This wavelength is also known asthe excitation wavelength. The energy absorbed by a flurochrome issubsequently released through various pathways, one being emission ofphotons to produce fluorescence. The wavelength of light being emittedis known as the emission wavelength and is an inherent characteristic ofa particular fluorophore. Radiationless energy transfer is thequantum-mechanical process by which the energy of the excited state ofone fluorophore is transferred without actual photon emission to asecond fluorophore. That energy may then be subsequently released at theemission wavelength of the second fluorophore. The first fluorophore isgenerally termed the donor (D) and has an excited state of higher energythan that of the second fluorophore, termed the acceptor (A). Theessential features of the process are that the emission specturm of thedonor overlap with the excitation spectrum of the acceptor, and that thedonor and acceptor be sufficiently close. The distance over whichradiationless energy transfer is effective depends on many factorsincluding the fluorescence quantum efficiency of the donor, theextinction coefficient of the acceptor, the degree of overlap of theirrespective spectra, the refractive index of the medium, and the relativeorientation of the transition moments of the two fluorophores. Inaddition to having an optimum emission range overlapping the excitationwavelength of the other fluorophore, the distance between D and A mustbe sufficiently small to allow the radiationless transfer of energybetween the fluorophores.

FRET may be performed either in vivo or in vitro. Proteins are labeledeither in vivo or in vitro by methods known in the art. According to theinvention, a natural binding domain, sequence or polypeptide and itsbinding partner, comprised either by the same or by differentpolypeptide molecules, are differentially labeled, one with a donor andthe other with an acceptor, and differences in fluorescence between atest assay, comprising a protein modifying enzyme, and a control, inwhich the modifying enzyme is absent, are measured using a fluorimeteror laser-scanning microscope. It will be apparent to those skilled inthe art that excitation/detection means can be augmented by theincorporation of photomultiplier means to enhance detection sensitivity.The differential labels may comprise either two different fluorescentlabels (e.g., fluorescent proteins as described below or thefluorophores rhodamine, fluorescein, SPQ, and others as are known in theart) or a fluorescent label and a molecule known to quench its signal;differences in the proximity of the natural binding domain, sequence orpolypeptide with its binding partner with and without theprotein-modifying enzyme can be gauged based upon a difference in thefluorescence spectrum or intensity observed.

This combination of protein-labeling methods and devices confers adistinct advantage over prior art methods for determining the activityof protein-modifying enzymes, as described above, in that results of allmeasurements are observed in real time (i.e., as a reaction progresses).This is significantly advantageous, as it allows both for rapid datacollection and yields information regarding reaction kinetics undervarious conditions.

A sample, whether in vitro or in vivo, assayed according to theinvention therefore comprises a mixture at equilibrium of the labelednatural binding domain, sequence or polypeptide and its binding partnerwhich, when disassociated from one another, fluoresce at one frequencyand, when complexed together, fluoresce at another frequency or,alternatively, of molecules which either do or do not fluoresce or showreduced fluorescence, depending upon whether or not they are associated.

The natural binding domain, sequence or polypeptide is modified to allowthe attachment of a fluorescent label to the surface of that molecule oris fused in-frame with a fluorescent protein, as described below. Thechoice of fluorescent label will be such that upon excitation withlight, labeled peptides which are associated will show optimal energytransfer between fluorophores. In the presence of a protein kinase orphosphatase, the natural binding domain, sequence or polypeptide and itsbinding partner dissociate due to a structural or electrostatic changewhich occurs as a consequence of addition or removal of a phosphateto/from the enzyme recognition site, thereby leading to a decrease inenergy transfer and increased emission of light by the donorfluorophore. In this way, the state of polypeptide phosphorylation canbe monitored and quantitated in real-time. This scheme, which representsthe broadest embodiment of the invention, is shown in FIG. 2.

As used herein, the terms “fluorophore” and “fluorochrome” referinterchangeably to a molecule which is capable of absorbing energy at awavelength range and releasing energy at a wavelength range other thanthe absorbance range. The term “excitation wavelength” refers to therange of wavelengths at which a fluorophore absorbs energy. The term“emission wavelength” refers to the range of wavelength that thefluorophore releases energy or fluoresces.

A non-limiting list of chemical fluorophores of use in the invention,along with their excitation and emission wavelengths, is presented inTable 1.

TABLE 1 Fluorophore Excitation (nm) Emission (nm) Color PKH2 490 504green PKH67 490 502 green Fluorescein (FITC) 495 525 green Hoechst 33258360 470 blue R-Phycoerythrin (PE) 488 578 orange-red Rhodamine (TRITC)552 570 red Quantum Red ™ 488 670 red PKH26 551 567 red Texas Red 596620 red Cy3 552 570 red

Examples of fluorescent proteins which vary among themselves inexcitation and emission maxima are listed in Table 1 of WO 97/28261(Tsien et al., 1997, supra). These (each followed by [excitationmax./emission max.] wavelengths expressed in nanometers) includewild-type Green Fluorescent Protein [395(475)/508] and the cloned mutantof Green Fluorescent Protein variants P4 [383/447], P4-3 [381/445], W7[433(453)/475(501)], W2 [432(453)/480], S65T [489/511], P4-1[504(396)/480], S65A [471/504], S65C [479/507], S65L [484/510], Y66F[360/442], Y66W [458/480], I0c [513/527], W1B [432(453)/476(503)],Emerald [487/508] and Sapphire [395/511]. This list is not exhaustive offluorescent proteins known in the art; additional examples are found inthe Genbank and SwissProt public databases.

A number of parameters of fluorescence output are envisaged including

1) measuring fluoresence emitted at the emission wavelength of theacceptor (A) and donor (D) and determining the extent of energy transferby the ratio of their emission amplitudes;

2) measuring the fluoresence lifetime of D;

3) measuring the rate of photobleaching of D;

4) measuring the anisotropy of D and/or A; or

5) measuring the Stokes shift monomer; excimer fluorescence. Certain ofthese techniques are presented below.

Alternative fluorescent techniques suitable for monitoringprotein:protein binding in assays of the invention

One embodiment of the technology can utilize monomer:excimerfluorescence as the output. The association of a natural binding domainwith a binding partner in this format is shown in FIG. 3.

The fluorophore pyrene when present as a single copy displaysfluorescent emission of a particular wavelength significantly shorterthan when two copies of pyrene form a planar dimer (excimer), asdepicted. As above, excitation at a single wavelength (probably 340 nm)is used to review the excimer fluorescence (˜470 nm) over monomerfluorescence (˜375 nm) to quantify assembly:disassembly of the reportermolecule.

Additional embodiments of the present invention are not dependent onFRET. For example the invention can make use of fluorescence correlationspectroscopy (FCS), which relies on the measurement of the rate ofdiffusion of a label (see Elson and Magde, 1974 Biopolymers, 13: 1-27;Rigler et al., 1992, in Fluorescence Spectroscopy: New Methods andApplications, Springer Verlag, pp.13-24; Eigen and Rigler, 1994, Proc.Natl. Acad. Sci. U.S.A., 91: 5740-5747; Kinjo and Rigler, 1995, NucleicAcids Res., 23: 1795-1799).

In FCS, a focused laser beam illuminates a very small volume ofsolution, of the order of 10⁻¹⁵ liter, which at any given point in timecontains only one molecule of the many under analysis. The diffusion ofsingle molecules through the illuminated volume, over time, results inbursts of fluorescent light as the labels of the molecules are excitedby the laser. Each individual burst, resulting from a single molecule,can be registered.

A labeled polypeptide will diffuse at a slower rate if it is large thanif it is small. Thus, multimerized polypeptides will display slowdiffusion rates, resulting in a lower number of fluorescent bursts inany given timeframe, while labeled polypeptides which are notmultimerized or which have dissociated from a multimer will diffuse morerapidly. Binding of polypeptides according to the invention can becalculated directly from the diffusion rates through the illuminatedvolume.

Where FCS is employed, rather than FRET, it is not necessary to labelmore than one polypeptide. Preferably, a single polypeptide member ofthe multimer is labeled. The labeled polypeptide dissociates from themultimer as a result of modification, thus altering the FCS reading forthe fluorescent label.

A further detection technique which may be employed in the method of thepresent invention is the measurement of time-dependent decay offluorescence anisotropy. This is described, for example, in Lacowicz,1983, Principles of Flourescence Spectroscopy, Plenum Press, New York,incorporated herein by reference (see, for example, page 167).

Fluorescence anisotropy relies on the measurement of the rotation offluorescent groups. Larger multimers of polypeptides rotate more slowlythan monomers, allowing the formation of multimers to be monitored.

Non-fluorescent detection methods for use in the invention

The invention may be configured to exploit a number of non-fluorescentlabels. In a first embodiment, the natural binding domain and bindingpartner therefor form, when bound, an active enzyme which is capable ofparticipating in an enzyme-substrate reaction which has a detectableendpoint. The enzyme may comprise two or more polypeptide chains orregions of a single chain, such that upon binding of the natural bindingdomain to the binding partner, which are present either on two differentpolypeptide chains or in two different regions of a single polypeptide,these components assemble to form a functional enzyme. Enzyme functionmay be assessed by a number of methods, including scintillation countingand photospectroscopy. In a further embodiment, the invention may beconfigured such that the label is a redox enzyme, for example glucoseoxidase, and the signal generated by the label is an electrical signal.

Phosphorylation of the natural binding domain and, optionally, itsbinding partner according to the invention is required to inhibitbinding and, consequently, enzyme component assembly, thus reducingenzyme activity.

In another assay format, an enzyme is used together with a modulator ofenzyme activity, such as an inhibitor or a cofactor. In such an assay,one of the enzyme and the inhibitor or cofactor is an natural bindingdomain, the other its binding partner. Binding of the enzyme to itsinhibitor or cofactor results in modulation of enzymatic activity, whichis detectable by conventional means (such as monitoring for theconversion of substrate to product for a given enzyme).

Fluorescent protein labels in assays of the invention

In a FRET assay of the invention, the fluorescent protein labels arechosen such that the excitation spectrum of one of the labels (theacceptor) overlaps with the emission spectrum of the excited fluorescentlabel (the donor). The donor label is excited by light of appropriateintensity within the donor's excitation spectrum. The donor then emitssome of the absorbed energy as fluorescent light and dissipates some ofthe energy by FRET to the acceptor fluorescent label. The fluorescentenergy it produces is quenched by the acceptor fluorescent proteinlabel. FRET can be manifested as a reduction in the intensity of thefluorescent signal from the donor, reduction in the lifetime of itsexcited state, and re-emission of fluorescent light at the longerwavelengths (lower energies) characteristic of the acceptor. When thedonor and acceptor labels become spatially separated, FRET is diminishedor eliminated.

One can take advantage of the FRET exhibited by a natural bindingdomain, sequence or polypeptide and its binding partner labeled withdifferent fluorescent proteins, wherein one is linked to a donor and theother to an acceptor fluorescent protein, in monitoring proteinphosphorylation according to the present invention. A single polypeptidemay comprises a blue fluorescent protein donor and a green fluorescentprotein acceptor, wherein each is fused to a different assay component(i.e., in which one is fused to the natural binding domain, sequence orpolypeptide and the other to its binding partner); such a construct isherein referred to as a “tandem” fusion protein. Alternatively, twodistinct polypeptides (“single” fusion proteins) one comprising anatural binding domain, sequence or polypeptide and the other itsbinding partner may be differentially labeled with the donor andacceptor fluorescent proteins, respectively. The construction and use oftandem fusion proteins in the invention can reduce significantly themolar concentration of peptides necessary to effect an associationbetween differentially-labeled polypeptide assay components relative tothat required when single fusion proteins are instead used. The labelednatural binding domain, sequence or polypeptide and/or its bindingpartner may be produced via the expression of recombinant nucleic acidmolecules comprising an in-frame fusion of sequences encoding a such apolypeptide and a fluorescent protein label either in vitro (e.g., usinga cell-free transcription/translation system, as described below, orinstead using cultured cells transformed or transfected using methodswell known in the art) or in vivo, for example in a transgenic animalincluding, but not limited to, insects, amphibians and mammals. Arecombinant nucleic acid molecule of use in the invention may beconstructed and expressed by molecular methods well known in the art,and may additionally comprise sequences including, but not limited to,those which encode a tag (e.g., a histidine tag) to enable easypurification, a secretion signal, a nuclear localization signal or otherprimary sequence signal capable of targeting the construct to aparticular cellular location, if it is so desired.

The means by which a natural binding domain, sequence or polypeptide andits binding partner are assayed for association using fluorescentprotein labels according to the invention may be briefly summarized asfollows:

Whether or not the natural binding domain, sequence or polypeptide andits binding partner are present on a single polypeptide molecule, one islabeled with a green fluorescent protein, while the other is preferablylabeled with a red or, alternatively, a blue fluorescent protein. Usefuldonor:acceptor pairs of fluorescent proteins (see Tsien et al., 1997,supra) include, but are not limited to:

Donor: S72A, K79R, Y145F, M153A and T203I (excitation λ 395 nm; emissionλ 511)

Acceptor: S65G, S72A, K79R and T203Y (excitation λ 514 nm; emission λ527 nm), or

T203Y/S65G, V68L, Q69K or S72A (excitation λ 515 nm; emission λ 527 nm).

An example of a blue:green pairing is P4-3 (shown in Table 1 of Tsien etal., 1997, supra) as the donor label and S65C (also of Table 1 of Tsienet al., 1997, supra) as the acceptor label. The natural binding domain,sequence or polypeptide and corresponding binding partner are exposed tolight at, for example, 368 nm, a wavelength that is near the excitationmaximum of P4-3. This wavelength excites S65C only minimally. Uponexcitation, some portion of the energy absorbed by the blue fluorescentprotein donor is transferred to the acceptor through FRET if the naturalbinding domain, sequence or polypeptide and its binding partner are inclose association. As a result of this quenching, the blue fluorescentlight emitted by the blue fluorescent protein is less bright than wouldbe expected if the blue fluorescent protein existed in isolation. Theacceptor (S65C) may re-emit the energy at longer wavelength, in thiscase, green fluorescent light.

After phosphorylation or dephosphorylation of one or both of the naturalbinding domain, sequence or polypeptide and its binding partner by ankinase or phosphatase, respectively, the natural binding domain,sequence or polypeptide and its binding partner (and, hence, the greenand red or, less preferably, green and blue fluorescent proteins)physically separate or associate, accordingly inhibiting or promotingFRET. For example, if activity of the modifying enzyme results indissociation of a protein:protein dimer, the intensity of visible bluefluorescent light emitted by the blue fluorescent protein increases,while the intensity of visible green light emitted by the greenfluorescent protein as a result of FRET, decreases.

Such a system is useful to monitor the activity of enzymes thatphosphorylate or dephosphorylate the phosphorylation site of a naturalbinding domain, sequence or polypeptide and, optionally, its bindingpartner to which the fluorescent protein labels are fused, as well asthe activity of kinases or phosphatases or candidate modulators of thoseenzymes.

In particular, this invention contemplates assays in which the amount-or activity of a modifying enzyme in a sample is determined bycontacting the sample with a natural binding domain, sequence orpolypeptide and its binding partner, differentially-labeled withfluorescent proteins, as described above, and measuring changes influorescence of the donor label, the acceptor label or the relativefluorescence of both. Fusion proteins, as described above, whichcomprise either one or both of the labeled natural binding domain,sequence or polypeptide and its binding partner of an assay of theinvention can be used for, among other things, monitoring the activityof a protein kinase or phosphatase inside the cell that expresses therecombinant tandem construct or two different recombinant constructs.

Advantages of single- and tandem fluorescent protein/polypeptidescomprising a natural binding domain, sequence or polypeptide fused to afluorescent protein include the potential to express the natural bindingdomain, sequence or polypeptide in the cell (providing a convenientexperimental format), the greater extinction coefficient and quantumyield of many of these proteins compared with those of the Edansfluorophore. Also, the acceptor in such a construct or pair ofconstructs is, itself, a fluorophore rather than a non-fluorescentquencher like Dabcyl. Alternatively, in single-label assays of theinvention, whether involving use of a chemical fluorophore or a singlefluorescent fusion construct, such a non-fluorescent quencher may beused. Thus, the enzyme's substrate (i.e., the natural binding domainand, optionally, the corresponding binding partner), and reactionproducts (i.e., the natural binding domain and, optionally, thecorresponding binding partner after modification) are both fluorescentbut with different fluorescent characteristics.

In particular, the substrate and modified products exhibit differentratios between the amount of light emitted by the donor and acceptorlabels. Therefore, the ratio between the two fluorescences measures thedegree of conversion of substrate to products, independent of theabsolute amount of either, the optical thickness of the sample, thebrightness of the excitation lamp, the sensitivity of the detector, etc.Furthermore, Aequorea-derived or -related fluorescent protein labelstend to be protease resistant. Therefore, they are likely to retaintheir fluorescent properties throughout the course of an experiment.

Reporter polyeptide fusion according to the invention

As stated above, recombinant nucleic acid constructs of particular usein the invention are those which comprise in-frame fusions of sequencesencoding a natural binding domain, sequence or polypeptide or a bindingpartner therefor and a fluorescent protein. If a natural binding domain,sequence or polypeptide and its binding partner are to be expressed aspart of a single polypeptide, the nucleic acid molecule additionallyencodes, at a minimum, a donor fluorescent protein fused to one, anacceptor fluorescent protein label fused to the other, a linker thatcouples the two and is of sufficient length and flexibility to allow forfolding of the polypeptide and pairing of the natural binding domain,sequence or polypeptide with the binding partner, and gene regulatorysequences operatively linked to the fusion coding sequence. If singlefusion proteins are instead encoded (whether by one or more nucleic acidmolecules), each nucleic acid molecule need only encode a naturalbinding domain, sequence or polypeptide or a binding partner therefor,fused either to a donor or acceptor fluorescent protein label andoperatively linked to gene regulatory sequences. “Operatively-linked”refers to polynucleotide sequences which are necessary to effect theexpression of coding and non-coding sequences to which they are ligated.The nature of such control sequences differs depending upon the hostorganism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

As described above, the donor fluorescent protein label is capable ofabsorbing a photon and transferring energy to another fluorescent label.The acceptor fluorescent protein label is capable of absorbing energyand emitting a photon. If needed, the linker connects the naturalbinding domain, sequence or polypeptide and its binding partner eitherdirectly or indirectly, through an intermediary linkage with one or bothof the donor and acceptor fluorescent protein labels. Regardless of therelative order of the natural binding domain, sequence or polypeptide,its binding partner and the donor and acceptor fluorescent proteinlabels on a polypeptide molecule, it is essential that sufficientdistance be placed between the donor and acceptor by the linker and/orthe natural binding domain, sequence or polypeptide and its bindingpartner to ensure that FRET does not occur unless the natural bindingdomain, sequence or polypeptide and its binding partner bind. It isdesirable, as described in greater detail in WO97/28261, to select adonor fluorescent protein with an emission spectrum that overlaps withthe excitation spectrum of an acceptor fluorescent protein. In someembodiments of the invention the overlap in emission and excitationspectra will facilitate FRET. Such an overlap is not necessary, however,if intrinsic fluorescence is measured instead of FRET. A fluorescentprotein of use in the invention includes, in addition to those withintrinsic fluorescent properties, proteins that fluoresce dueintramolecular rearrangements or the addition of cofactors that promotefluorescence.

For example, green fluorescent proteins (“GFPs”) of cnidarians, whichact as their energy-transfer acceptors in bioluminescence, can be usedin the invention. A green fluorescent protein, as used herein, is aprotein that fluoresces green light, and a blue fluorescent protein is aprotein that fluoresces blue light. GFPs have been isolated from thePacific Northwest jellyfish, Aequorea victoria, from the sea pansy,Renilla reniformis, and from Phialidium gregarium. (Ward et al., 1982,Photochem. Photobiol., 35: 803-808; Levine et al., 1982, Comp. Biochem.Physiol., 72B: 77-85).

A variety of Aequorea-related GFPs having useful excitation and emissionspectra have been engineered by modifying the amino acid sequence of anaturally occurring GFP from Aequorea victoria. (Prasher et al., 1992,Gene, 111: 229-233; Heim et al., 1994, Proc. Natl. Acad. Sci. U.S.A.,91: 12501-12504; PCT/US95/14692). As used herein, a fluorescent proteinis an Aequorea-related fluorescent protein if any contiguous sequence of150 amino acids of the fluorescent protein has at least 85% sequenceidentity with an amino acid sequence, either contiguous ornon-contiguous, from the wild-type Aequorea green fluorescent protein ofSwissProt Accession No. P42212. Similarly, the fluorescent protein maybe related to Renilla or Phialidium wild-type fluorescent proteins usingthe same standards.

Aequorea-related fluorescent proteins include, for example, wild-type(native) Aequorea victoria GFP, whose nucleotide and deduced amino acidsequences are presented in Genbank Accession Nos. L29345, M62654, M62653and others Aequorea-related engineered versions of Green FluorescentProtein, of which some are listed above. Several of these, i.e., P4,P4-3, W7 and W2 fluoresce at a distinctly shorter wavelength than wildtype.

Recombinant nucleic acid molecules encoding single- or tandemfluorescent protein/polypeptide comprising a natural binding domain,sequence or polypeptide or a binding partner therefor fused to afluorescent protein useful in the invention may be expressed for in vivoassay of the activity of a modifying enzyme on the encoded products.Alternatively, the encoded fusion proteins may be isolated prior toassay, and instead assayed in a cell-free in vitro assay system, asdescribed elsewhere herein.

Protein phosphorylation in assays of the invention

As highlighted in the Background, the phosphorylation of proteins is afrequent and important post-translational modification of proteins.There are many examples of situations in which dysfunction of thekinases and phosphatases mediating the phosphorylation state of proteinscan lead to disease. The methods currently available to analyze thephosphorylation state each have drawbacks, as described above. Assayformats of the invention, as outlined in the following sections and inthe Examples, below, will allow monitoring of the phosphorylation stateof a specific target protein or activity of a specific kinase orphosphatase in real time in the cell.

Three systems, presented in Examples 1 through 4, can be used toexemplify in non-limiting fashion the phosphorylation assay, each ofwhich involves the interaction between a binding domain, sequence orpolypeptide and a binding partner, of which at least the formercomprises a modification site that serves as a substrate for the proteinkinases and phosphatases involved in the system. At the present time,good structural information is available for such interactions.

Methods for detection of protein phosphorylation in real time

A. A in vitro protein modification and detection thereof

Modifying enzymes

The invention requires the presence of a modifying enzyme whichcatalyzes either the addition or removal of a modifying group. A rangeof kinases, phosphatases and other modifying enzymes are availablecommercially (e.g. from Sigma, St. Louis, Mo.; Promega, Madison, Wis.;Boehringer Mannheim Biochemicals, Indianapolis, Ind.; New EnglandBiolabs, Beverly, Mass.; and others). Alternatively, such enzymes may beprepared in the laboratory by methods well known in the art.

The catalytic sub-unit of protein kinase A (c-PKA) can be purified fromnatural sources (e.g. bovine heart) or from cells/organisms engineeredto heterologously express the enzyme. Other isofonrns of this enzyme maybe obtained by these procedures. Purification is performed as previouslydescribed from bovine heart (Peters et al.,1977, Biochemistry, 16:5691-5697) or from a heterologous source (Tsien et al., WO92/00388), andis in each case briefly summarized as follows:

Bovine ventricular cardiac muscle (2 kg) is homogenized and thencentrifuged. The supernatant is applied to a strong anion exchange resin(e.g. Q resin, Bio-Rad) equilibrated in a buffer containing 50 mMTris-HCl, 10 mM NaCl, 4 mM EDTA pH 7.6 and 0.2 mM 2-mercaptoethanol. Theprotein is eluted from the resin in a second buffer containing 50 mMTris-HCI, 4 mM EDTA pH 7.6, 0.2 mM 2-mercaptoethanol, 0.5M NaCi.Fractions containing PKA are pooled and ammonium sulphate added to 30%saturation. Proteins precipitated by this are removed by centrifugationand the ammonium sulphate concentration of the supernatant was increasedto 75% saturation. Insoluble proteins are collected by centrifugation(included c-PKA) and are dissolved in 30 mM phosphate buffer pH 7.0, 1mM EDTA, 0.2 mM 2-mercaptoethanol. These proteins are then dialysedagainst the same buffer (500 volume excess) at 4° C for two periods of 8hours each. The pH of the sample is reduced to 6.1 by addition ofphosphoric acid, and the sample is mixed sequentially with 5 batches ofCM-Sepharose (Pharmacia, ˜80 ml resin each) equilibrated in 30 mMphosphate pH 6.1, 1 mM EDTA, 0.2 mM 2-mercaptoethanol. Cyclic AMP (10μM) is added to the material which fails to bind to the CM-Sepharose,and the sample- cAMP mix is incubated with a fresh resin of CM-Sepharose(˜100 ml) equilibrated as before. c-PKA is eluted from this columnfollowing extensive washing in equilibration buffer by addition of 3OmMphosphate pH 6.1, 1 mM EDTA, IM KCI, 0.2 mM 2- mercaptoethanol.Fractions containing c-PKA are pooled and concentrated by filtrationthrough a PM-30 membrane (or similar). The c-PKA sample is thensubjected to gel- filtration chromatography on a resin such as Sephacryl200HR (Pharmacia).

The purification of recombinant c-PKA is as described in WO 92/00388.General methods of preparing pure and partially-purified recombinantproteins, as well as crude cellular extracts comprising such proteins,are well known in the art. Molecular methods useful in the production ofrecombinant proteins, whether such proteins are the enzymes to beassayed according to the invention or the labeled reporter polypeptidesof the invention (i.e., the natural binding domain, sequence orpolypeptide and its binding partner), are well known in the art (formethods of cloning, expression of cloned genes and protein purification,see Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual., 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Ausubel et al., Current Protocols in Molecular Biology, copyright1987-1994, Current Protocols, copyright 1994-1998, John Wiley & Sons,Inc.). The sequences of the catalytic subunit of several PKA moleculesare found in the Genbank database (see PKA Cα, bovine, Genbank AccessionNos. X67154 and S49260; PKA Cβ1, bovine, Genbank Accession No. J02647;PKA Cβ2, bovine, M60482, the form most likely purified from bovine heartby the protocol described above).

According to the invention, assays of the activity of protein kinases orphosphatases may be performed using crude cellular extracts, whether totest the activity of a recombinant protein or one which is found innature, such as in a biological sample obtained from a test cell line oranimal or from a clinical patient. In the former case, use of a crudecell extract enables rapid screening of many samples, which potentiallyfinds special application in high-throughput screening methods, e.g. ofcandidate modulators of protein kinase/phosphatase activity. In thelatter case, use of a crude extract with the labeled reporterpolypeptide comprising a natural binding domain, sequence or polypeptideof the invention facilitates easy and rapid assessment of the activityof an enzyme of interest in a diagnostic procedure, e.g., one which isdirected at determining whether a protein kinase or phosphatase isactive at an a physiologically-appropriate level, or in a proceduredesigned to assess the efficacy of a therapy aimed at modulating theactivity of a particular enzyme.

Production of a natural binding domain, sequence or polypeptide

Polypeptides comprising or consisting of a natural binding domain,sequence or polypeptide or a binding partner thereof may be synthesizedby Fmoc or Tboc chemistry according to methods known in the art (e.g.,see Atherton et al., 1981, J. Chem. Soc. Perkin I, 1981(2): 538-546;Merrifield, 1963, J. Am. Chem. Soc., 85: 2149-2154, respectively).Following deprotection and cleavage from the resin, peptides aredesalted by gel filtration chromatography and analysed by massspectroscopy, HPLC, Edman degradation and/or other methods as are knownin the art for protein sequencing using standard methodologies.

Alternatively, nucleic acid sequences encoding such peptides may beexpressed either in cells or in an in vitro transcription/translationsystem (see below) and, as with enzymes to be assayed according to theinvention, the proteins purified by methods well known in the art.

Labelling of polypeptides with fluorophores

Polypeptides comprising or consisting of natural binding domains,sequences or polypeptides or a binding partner therefor are labeled withthiol reactive derivatives of fluorescein and tetramethylrhodamine(isothiocyanate or iodoacetamide derivatives, Molecular Probes, Eugene,OR, USA) or other fluorophores as are known in the art using proceduresdescribed by Hermanson G.T., 1995, Bioconjugate ues, Academic Press,London. Alternatively, primary-amine-directed conjugation reactions canbe used to label lysine sidechains or the free peptide N-terminus(Hermason, 1995, supra).

Purification of fluorescent natural binding domains and/or bindingpartners therefor

Fluorescent peptides are separated from unreacted fluorophores by gelfiltration chromatography or reverse phase HPLC.

Phosphorylation of natural binding domains and, optionally, bindingpartners therefor in vitro

Natural binding domains and, optionally, binding partners therefor(0.01-100.0CM) are phosphorylated by purified c-PKA in 5OmM Histidinebuffer pH 7.0, 5 mM MgSO₄, I lmM EGTA, 0.1-10.0 μM c-PKA, and 0.2 mM[³²P] γ-ATP (specific activity ˜2Bq/pmol) at 15-40° C. for periods oftime ranging from 0 to 60 minutes. Where the chemistry of the peptide isappropriate (i.e. having a basic charge) the phosphopeptide is capturedon a cation exchange filter paper (e.g. phosphocellulose P81 paper;Whatman), and reactants are removed by extensive washing in 1%phosphoric acid (see Casnellie, 1991, Methods Enzynmol., 200: 115-120).Alternatively, phosphorylation of samples is terminated by the additionof SDS-sample buffer (Laemmli,1970, Nature, 227: 680-685) and thesamples analysed by SDS-PAGE electrophoresis, autoradiography andscintillation counting of gel pieces.

Dephosphorylation of a natural binding domain or binding partnertherefor in vitro

The dephosphorylation of natural binding domains and, optionally,binding partners therefor, phosphorylated as above is studied by removalof ATP (through the addition of 10 mM glucose and 30 U/ml hexokinase;Sigma, St. Louis, Mo.) and addition of protein phosphatase-1 (Sigma).Dephosphorylation is followed at 15-40° C. by quantitation of theremaining phosphopeptide component at various time points, determined asabove.

Fluorescence measurements of protein modification in vitro in real time

Donor and acceptor fluorophore-labeled polypeptides comprising orconsisting of natural binding domains, sequences or polypeptides (molarequivalents of fluorophore- labeled polypeptide or molar excess ofacceptor-labeled polypeptide) are first mixed (if the natural bindingdomains, sequence or polypeptide and its binding partner are present onseparate polypeptides). Samples are analyzed in a fluorimeter usingexcitation wavelengths relevant to the donor fluorescent label andemission wavelengths relevant to both the donor and acceptor labels. Aratio of emission from the acceptor over that from the donor followingexcitation at a single wavelength is used to determine the efficiency offluorescence energy transfer between fluorophores, and hence theirspatial proximity. Typically, measurements are performed at 0-37° C as afunction of time following the addition of the modifying enzyme (and,optionally, a modulator or candidate modulator of function for thatenzyme, as described below) to the system in 5OmM histidine pH 7.0, 120mM KCl, 5 mM MgSO₄, 5 mM NaF, 0.05 mM EGTA and 0.2 mM ATP. The assay maybe performed at a higher temperature if that temperature is compatiblewith the enzyme(s) under study.

Altenative cell-free assay system of the invention

A cell-free assay system according to the invention is required topermit binding of an unmodified, labeled natural binding domain,sequence or polypeptide and its binding partner to occur. As indicatedherein, such a system may comprise a low-ionic-strength buffer (e.g.,physiological salt, such as simple saline or phosphate- and/orTris-buffered saline or other as described above), a cell culturemedium, of which many are known in the art, or a whole or fractionatedcell lysate. The components of an assay of protein modificationaccording to the invention may be added into a buffer, medium or lysateor may have been expressed in cells from which a lysate is derived.Alternatively, a cell-free transcription- and/or translation system maybe used to deliver one or more of these components to the assay system.Nucleic acids of use in cell-free expression systems according to theinvention are as described for in vivo assays, below.

An assay of the invention may be peformed in a standard in vitrotranscription/translation system under conditions which permitexpression of a recombinant or other gene. The TNT® T7 Quick CoupledTranscription/Translation System (Cat.# L1170; Promega) contains allreagents necessary for in vitro transcription/translation except the DNAof interest and the detection label; as discussed below, polypeptidescomprising natural binding domains, sequences or polypeptides or theirbinding partners may be encoded by expression constructs in which theircoding sequences are fused in-frame to those encoding fluorescentproteins. The TNT® Coupled Reticulocyte Lysate Systems (comprising arabbit reticulocyte lysate) include: TNT®V T3 Coupled ReticulocyteLysate System (Cat. # L4950; Promega); TNT® T7 Coupled ReticulocyteLysate System (Cat. # L4610; Promega); TNT® SP6 Coupled ReticulocyteLysate System (Cat. # L4600; Promega); TNT® T7/SP6 Coupled ReticulocyteLysate System (Cat. # L5020; Promega); TNT® T7/T3 Coupled ReticulocyteLysate System (Cat. # L5010; Promega).

An assay involving a cell lysate or a whole cell (see below) may beperformed in a cell lysate or whole cell preferably eukaryotic in nature(such as yeast, fungi, insect, e.g., Drosophila), mouse, or human). Anassay in which a cell lysate is used is performed in a standard in vitrosystem under conditions which permit gene expression. A rabbitreticulocyte lysate alone is also available from Promega, eithernuclease-treated (Cat. # L4960) or untreated (Cat. # L4151).

Candidate modulators is of protein kinases and/or phosphatases to bescreened according to the invention

Whether in vitro or in an in vivo system (see below), the inventionencompasses methods by which to screen compositions which may enhance,inhibit or not affect (e.g., in a cross-screening procedure in which thegoal is to determine whether an agent intended for one purposeadditionally affects general cellular functions, of which proteinphosphorylation/dephosphorylation is an example) the activity of aprotein kinase or phosphatase.

Candidate modulator compounds from large libraries of synthetic ornatural compounds can be screened. Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds. Synthetic compound libraries are commercially availablefrom a number of companies including Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich (Milwaukee, Wis.). Combinatoriallibraries are available and can be prepared. Alternatively, libraries ofnatural compounds in the form of bacterial, fungal, plant and animalextracts are available from e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (NC), or are readily produceable by methods well known in theart. Additionally, natural and synthetically produced libraries andcompounds are readily modified through conventional chemical, physical,and biochemical means.

Useful compounds may be found within numerous chemical classes, thoughtypically they are organic compounds, including small organic compounds.Small organic compounds have a molecular weight of more than 50 yet lessthan about 2,500 daltons, preferably less than about 750, morepreferably less than about 350 daltons. Exemplary classes includeheterocycles, peptides, saccharides, steroids, and the like. Thecompounds may be modified to enhance efficacy, stability, pharmaceuticalcompatibility, and the like. Structural identification of an agent maybe used to identify, generate, or screen additional agents. For example,where peptide agents are identified, they may be modified in a varietyof ways to enhance their stability, such as using an unnatural aminoacid, such as a D-amino acid, particularly D-alanine, by functionalizingthe amino or carboxylic terminus, e.g. for the amino group, acylation oralkylation, and for the carboxyl group, esterification or amidification,or the like.

Candidate modulators which may be screened according to the methods ofthe invention include receptors, enzymes, ligands, regulatory factors,and structural proteins. Candidate modulators also include nuclearproteins, cytoplasmic proteins, mitochondrial proteins, secretedproteins, plasmalemma-associated proteins, serum proteins, viralantigens, bacterial antigens, protozoal antigens and parasitic antigens.Candidate modulators additionally comprise proteins, lipoproteins,glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such asribozymes or antisense nucleic acids). Proteins or polypeptides whichcan be screened using the methods of the present invention includehormones, growth factors, neurotransmitters, enzymes, clotting factors,apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumorsuppressors, structural proteins, viral antigens, parasitic antigens,bacterial antigens and antibodies (see below).

Candidate modulators which may be screened according to the inventionalso include substances for which a test cell or organism might bedeficient or that might be clinically effective in higher-than-normalconcentration as well as those that are designed to eliminate thetranslation of unwanted proteins. Nucleic acids of use according to theinvention not only may encode the candidate modulators described above,but may eliminate or encode products which eliminate deleteriousproteins. Such nucleic acid sequences are antisense RNA and ribozymes,as well as DNA expression constructs that encode them. Note thatantisense RNA molecules, ribozymes or genes encoding them may beadministered to a test cell or organism by a method of nucleic aciddelivery that is known in the art, as described below. Inactivatingnucleic acid sequences may encode a ribozyme or antisense RNA specificfor the a target MRNA. Ribozymes of the hammerhead class are thesmallest known, and lend themselves both to in vitro production anddelivery to cells (summarized by Sullivan, 1994, J. Invest. Dermatol.,103: 85S-98S; Usman et al., 1996, Curr. Opin. Struct. Biol., 6:527-533).

As stated above, antibodies are of use in the invention as modulators(specifically, as inhibitors) of protein kinases and/or phosphatases.Methods for the preparation of antibodies are well known in the art, andare briefly summarized as follows:

Either recombinant proteins or those derived from natural sources can beused to generate antibodies using standard techniques, well known tothose in the field. For example, the proteins are administered tochallenge a mammal such as a monkey, goat, rabbit or mouse. Theresulting antibodies can be collected as polyclonal sera, orantibody-producing cells from the challenged animal can be immortalized(e.g. by fusion with an immortalizing fusion partner) to producemonoclonal antibodies.

1. Polyclonal antibodies.

The antigen protein may be conjugated to a conventional carrier in orderto increases its immunogenicity, and an antiserum to the peptide-carrierconjugate is raised. Coupling of a peptide to a carrier protein andimmunizations may be performed as described (Dymecki et al., 1992, J.Biol. Chem., 267: 4815-4823). The serum is titered against proteinantigen by ELISA (below) or alternatively by dot or spot blotting(Boersma and Van Leeuwen, 1994, J. Neurosci. Methods, 51: 317). At thesame time, the antiserum may be used in tissue sections prepared asdescribed below. The serum is shown to react strongly with theappropriate peptides by ELISA, for example, following the procedures ofGreen et al., 1982, Cell, 28: 477-487.

2. Monoclonal antibodies.

Techniques for preparing monoclonal antibodies are well known, andmonoclonal antibodies may be prepared using a candidate antigen whoselevel is to be measured or which is to be either inactivated oraffinity-purified, preferably bound to a carrier, as described byAmheiter et al., Nature, 294, 278-280 (1981).

Monoclonal antibodies are typically obtained from hybridoma tissuecultures or from ascites fluid obtained from animals into which thehybridoma tissue is introduced. Nevertheless, monoclonal antibodies maybe described as being “raised to” or “induced by” a protein.

Monoclonal antibody-producing hybridomas (or polyclonal sera) can bescreened for antibody binding to the target protein. By antibody, weinclude constructions using the binding (variable) region of such anantibody, and other antibody modifications. Thus, an antibody useful inthe invention may comprise a whole antibody, an antibody fragment, apolyfunctional antibody aggregate, or in general a substance comprisingone or more specific binding sites from an antibody. The antibodyfragment may be a fragment such as an Fv, Fab or F(ab′)₂ fragment or aderivative thereof, such as a single chain Fv fragment. The antibody orantibody fragment may be non-recombinant, recombinant or humanized. Theantibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and soforth. In addition, an aggregate, polymer, derivative and conjugate ofan immunoglobulin or a fragment thereof can be used where appropriate.

Determination of activity of candidate modulator of a protein kinase orphosphatase

A candidate modulator of the activity of a protein kinase or phosphatasemay be assayed according to the invention as described herein, isdetermined to be effective if its use results in a difference of about10% or greater relative to controls in which it is not present (seebelow) in FRET resulting from the association of a labeled naturalbinding domain, sequence or polypeptide and its binding partner in thepresence of a protein-modifying enzyme.

The level of activity of a candidate modulator may be quantified usingany acceptable limits, for example, via the following formula:${{Percent}\quad {Modulation}} = {\frac{\left( {{Index}_{Control} - {Index}_{Sample}} \right)}{\left( {Index}_{Control} \right)} \times 100}$

where Index_(Control) is the quantitative result (e.g., amount of- orrate of change in fluorescence at a given frequency, rate of molecularrotation, FRET, rate of change in FRET or other index of modification,including, but not limited to, enzyme inhibition or activation) obtainedin assays that lack the candidate modulator (in other words, untreatedcontrols), and Indexsample represents the result of the same measurementin assays containing the candidate modulator. As described below,control measurements are made with differentially labeled naturalbinding domains, sequences or polypeptides and their binding partnersonly, and then with these molecules plus a protein kinase or phosphatasewhich recognizes a phosphorylation site present on them.

Such a calculation is used in either in vitro or in vivo assaysperformed according to the invention.

B. In vivo assays of enzymatic activity according to the invention

Reporter group protein modification in living cells

Differentially-labeled natural binding domains, sequences orpolypeptides and their corresponding binding partners of the inventionare delivered (e.g., by microinjection) to cells, such as smooth musclecells (DDTI) or ventricular cardiac myocytes as previously described(Riabowol et al., 1988, Cold Spring Harbor Symposia on QuantitatveBiology, 53: 85-90). The ratio of emission from the labeled molecule(s)is measured as described above via a photomultiplier tube focused on asingle cell. Activation of a kinase (e.g., PKA by the addition ofdibutyryl cAMP or P-adrenergic agonists) is performed, subsequentinhibition is performed by removal of stimulus and by addition of asuitable antagonist (e.g., cAMP antagonist Rp-cAMPS).

Heterologous expression of peptides

Natural binding domains, sequences or polypeptides and/or their bindingpartners can be produced from the heterologous expression of DNAsequences that encode them or by chemical synthesis of the same.Expression can be in procaryotic or eukaryotic cells using a variety ofplasmid vectors capable of instructing heterologous expression.Purification of these products is achieved by destruction of the cells(e.g. French Press) and chromatographic purification of the products.This latter procedure can be simplified by the inclusion of an affinitypurification tag at one extreme of the peptide, separated from thepeptide by a protease cleavage site if necessary.

The use of cells or whole organisms in assays of the invention

When performed using cells, the assays of the invention are broadlyapplicable to a host cell susceptible to transfection or transformationincluding, but not limited to, bacteria (both gram-positive andgram-negative), cultured- or explanted plant (including, but not limitedto, tobacco, arabidopsis, carnation, rice and lentil cells orprotoplasts), insect (e.g., cultured Drosophila or moth cell lines) orvertebrate cells (e.g., mammalian cells) and yeast.

Organisms are currently being developed for the expression of agentsincluding DNA, RNA, proteins, non-proteinaceous compounds, and viruses.Such vector microorganisms include bacteria such as Clostridium (Parkeret al., 1947, Proc. Soc. Exp. Biol. Med., 66: 461-465; Fox et al., 1996,GeneTherapy, 3: 173-178; Minton et al., 1995, FEMS Microbiol. Rev., 17:357-364), Salmonella (Pawelek et al., 1997, Cancer Res., 57: 4537-4544;Saltzman et al., 1996, Cancer Biother. Radiopharm., 11: 145-153; Carrieret al., 1992, J. Immunol., 148: 1176-1181; Su et al., 1992, Microbiol.Pathol., 13: 465-476; Chabalgoity et al., 1996, Infect. Immunol., 65:2402-2412), Listeria (Schafer et al., 1992, J. Immunol., 149: 53-59; Panet al., 1995, Nature Med., 1: 471-477) and Shigella (Sizemore et al.,1995, Science, 270: 299-302), as well as yeast, mycobacteria, slimemolds (members of the taxa Dictyosteliida—such as of the generaPolysphondylium and Dictystelium, e.g. Dictyostelium discoideum—andMyxomycetes—e.g. of the genera Physarum and Didymium) and members of theDomain Arachaea (including, but not limited to, archaebacteria), whichhave begun to be used in recombinant nucleic acid work, members of thephylum Protista, or other cell of the algae, fungi, or any cell of theanimal or plant kingdoms.

Plant cells useful in expressing polypeptides of use in assays of theinvention include, but are not limited to, tobacco (Nicotianaplumbaginifolia and Nicotiana tabacum), arabidopsis (Arabidopsisthaliana), Aspergillus niger, Brassica napus, Brassica nigra, Daturainnoxia, Vicia narbonensis, Viciafaba, pea (Pisum sativum), cauliflower,carnation and lentil (Lens culinaris). Either whole plants, cells orprotoplasts may be transfected with a nucleic acid of choice. Methodsfor plant cell transfection or stable transformation include inoculationwith Agrobacterium tumefaciens cells carrying the construct of interest(see, among others, Turpen et al., 1993, I-.VLirol.Mitllds, 42: 227-239), administration of liposome-associated nucleic acid molecules(Maccarrone et al., 1992, Biochem.BiophysRes.Corinun., 186: 1417-1422)and microparticle injection (Johnston and Tang, 1993, Genet. Eng. (NY),15: 225-236), among other methods. A generally useful planttranscriptional control element is the cauliflower mosaic virus (CaMV)35S promoter (see, for example, Saalbach et al., 1994, Mol. Gen. Genet.,242: 226-236). Non-limiting examples of nucleic acid vectors useful inplants include pGSGLUCl (Saalbach et al., 1994, supra), pGA492 (Perez etal., 1989, Plant. Mol. Biol., 13: 365-373), pOCA18 (Olszewski et al.,1988, Nucleic. Acids Res., 16: 10765-10782), the Ti plasmid (Roussell etal., 1988, Mol. Gen. Genet., 211: 202-209) and pKR612B1 (Balazs et al.,1985, Gene, 40: 343-348).

Mammalian cells are of use in the invention. Such cells include, but arenot limited to, neuronal cells (those of both primary explants and ofestablished cell culture lines) cells of the immune system (such asT-cells, B-cells and macrophages), fibroblasts, hematopoietic cells anddendritic cells. Using established technologies, stem cells (e.g.hematopoietic stem cells) may be used for gene transfer after enrichmentprocedures. Alternatively, unseparated hematopoietic cells and stem cellpopulations may be made susceptible to DNA uptake. Transfection ofhematopoietic stem cells is described in Mannion-Henderson et al., 1995,Exp. Hemato., 23: 1628; Schiffmann et al., 1995, Blood, 86: 1218 ;Williams, 1990, Bone Marrow Transplant, 5: 141; Boggs, 1990, Int. J.Cell Cloning, 8: 80; Martensson et al., 1987, Eur. J. Immunol,. 17:1499; Okabe et al., 1992, Eur. J. Immunol., 22: 37-43; and Banerji etal., 1983, Cell, 33: 729. Such methods may advantageously be usedaccording to the present invention.

Nucleic acid vectors for the expression of assay components of theinvention in cells or multicellular organisms

A nucleic acid of use according to the methods of the invention may beeither double- or single stranded and either naked or associated withprotein, carbohydrate, proteoglycan and/or lipid or other molecules.Such vectors may contain modified and/or unmodified nucleotides orribonucleotides. In the event that the gene to be transfected may bewithout its native transcriptional regulatory sequences, the vector mustprovide such sequences to the gene, so that it can be expressed onceinside the target cell. Such sequences may direct transcription in atissue-specific manner, thereby limiting expression of the gene to itstarget cell population, even if it is taken up by other surroundingcells. Alternatively, such sequences may be general regulators oftranscription, such as those that regulate housekeeping genes, whichwill allow for expression of the transfected gene in more than one celltype; this assumes that the majority of vector molecules will associatepreferentially with the cells of the tissue into which they wereinjected, and that leakage of the vector into other cell types will notbe significantly deleterious to the recipient organism. It is alsopossible to design a vector that will express the gene of choice in theTarget cells at a specific time, by using an inducible promoter, whichwill not direct transcription unless a specific stimulus, such as heatshock, is applied.

A gene encoding a component of the assay system of the invention or acandidate modulator of protein kinase or phosphatase activity may betransfected into a cell or organism using a viral or non-viral DNA orRNA vector, where non-viral vectors include, but are not limited to,plasmids, linear nucleic acid molecules, artificial chromomosomes andepisomal vectors. Expression of heterologous genes in mammals has beenobserved after injection of plasmid DNA into muscle (Wolff J. A. et al.,1990, Science, 247: 1465-1468; Carson D. A. et al., U.S. Pat. No.5,580,859), thyroid (Sykes et al. 1994, Human Gene Ther., 5: 837-844),melanoma (Vile et al., 1993, Cancer Res., 53: 962-967), skin (Hengge etal., 1995, Nature Genet., 10: 161-166), liver (Hickman et al., 1994,Human Gene Therapy, 5: 1477-1483) and after exposure of airwayepithelium (Meyer et al., 1995, Gene Therapy, 2: 450-460).

In addition to vectors of the broad classes described above and fusiongene expression construct encoding a natural binding domain, sequence orpolypeptide fused in-frame to a fluorescent protein, as described above(see “Fluorescent resonance energy transfer”), microbial plasmids, suchas those of bacteria and yeast, are of use in the invention.

Bacterial plasmids:

Of the frequently used origins of replication, pBR322 is usefulaccording to the invention, and pUC is preferred. Although notpreferred, other plasmids which are useful according to the inventionare those which require the presence of plasmid encoded proteins forreplication, for example, those comprising pT181, FII, and Fl origins ofreplication.

Examples of origins of replication which are useful in assays of theinvention in E. coli and S. typhimurium include but are not limited to,pHETK (Garapin et al., 1981, Proc. Natl. Acad. Sci U.S.A, 78: 815-819),p279 (Talmadge et al., 1980, Proc. Natl. Acad. Sci. U.SA., 77:3369-3373), p5-3 and p21A-2 (both from Pawalek et al., 1997, CancerRes., 57: 4537-4544), pMB1 (Bolivar et al., 1977, Gene, 2: 95-113),ColEl (Kahn et al., 1979, Methods Enzymol., 68: 268-280), p15A (Chang etal., 1978, J. Bacteriol, 134: 1141-1156); pSC101 (Stoker et al., 1982,Gene, 18: 335-341); R6K (Kahn et al., 1979, supra); R1 (temperaturedependent origin of replication, Uhlin et al., 1983, Gene, 22: 255-265);lambda dv (Jackson et al., 1972, Proc. Nat. Aca. Sci. U.S.A., 69:2904-2909); pYA (Nakayama et al., 1988, infra). An example of an originof replication that is useful in Staphylococcus is pT181 (Scott, 1984,Microbial Reviews 48: 1-23). Of the above-described origins ofreplication, pMB 1, p15A and ColEI are preferred because these originsdo not require plasmid-encoded proteins for replication.

Yeast plasmids:

Three systems are used for recombinant plasmid expression andreplication in yeasts:

1. Integrating. An example of such a plasmid is YIp, which is maintainedat one copyper haploid genome, and is inherited in Mendelian fashion.Such a plasmid, containing a gene of interest, a bacterial origin ofreplication and a selectable gene (typically an antibiotic-resistancemarker), is produced in bacteria. The purified vector is linearizedwithin the selectable gene and used to transform competent yeast cells.Regardless of the type of plasmid used, yeast cells are typicallytransformed by chemical methods (e.g. as described by Rose et al., 1990,Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). The cells are treated with lithium acetate toachieve transformation efficiencies of approximately 10⁴ colony-formingunits (transformed cells)/μg of DNA. Yeast perform homologousrecombination such that the cut, selectable marker recombines with themutated (usually a point mutation or a small deletion) host gene torestore function. Transformed cells are then isolated on selectivemedia.

2. Low copy-number ARS-CEN, of which YCp is an example. Such a plasmidcontains the autonomous replicating sequence (ARS1), a sequence ofapproximately 700 bp which, when carried on a plasmid, permits itsreplication in yeast, and a centromeric sequence (CEN4), the latter ofwhich allows mitotic stability. These are usually present at 1-2 copiesper cell. Removal of the CEN sequence yields a YRp plasmid, which istypically present in 100-200 copes per cell; however, this plasmid isboth mitotically and meioticaily unstable.

3. High-copy-number 2 μ circles. These plasmids contain a sequenceapproximately 1 kb in length, the 2μ sequence, which acts as a yeastreplicon giving rise to higher plasmid copy number; however, theseplasmids are unstable and require selection for maintenance. Copy numberis increased by having on the plasmid a selection gene operativelylinked to a crippled promoter. This is usually the LEU2 gene with atruncated promoter (LEU2-d), such that low levels of the Leu2p proteinare produced; therefore, selection on a leucine-depleted medium forcesan increase in copy number in order to make an amount of Leu2psufficient for cell growth.

As suggested above, examples of yeast plasmids use fill in the inventioninclude the YRp plasmids (based on autonomously-replicating sequences,or ARS) and the YEp plasmids (based on the 2 μ circle), of whichexamples are YEp24 and the YEplac series of plasmids (Gietz and Sugino,1988, Gene, 74: 527-534). (See Sikorski, “Extrachromosomal cloningvectors of Saccharomyces cerevisiae”, in Plasmids, A Practical Approach,Ed. K. G. Hardy, IRL Press, 1993; and Yeast Clonig Vectors and GenesCurrent Protocols in Molecular Biology, Section II, Unit 13.4, Eds.,Ausubel et al., 1994).

In addition to a yeast origin of replication, yeast plasmid sequencestypically comprise an antibiotic resistance gene, a bacterial origin ofreplication (for propagation in bacterial cells) and a yeast nutritionalgene for maintenance in yeast cells. The nutritional gene (or“auxotrophic marker”) is most often one of the following (with the geneproduct listed in parentheses and the sizes quoted encompassing thecoding sequence, together with the promoter and terminator elementsrequired for correct expression):

TRP1 (PhosphoADP-ribosylanthranilate isomerase, which is a component ofthe tryptophan biosynthetic pathway).

URA3 (Orotidine-5′-phosphate decarboxylase, which takes part in theuracil biosynthetic pathway).

LEU2 (3-Isopropylmalate dehydrogenase, which is involved with theleucine biosynthetic pathway).

HIS3 (Imidazoleglycerolphosphate dehydratase, or IGP dehydratase).

LYS2 (α-aminoadipate-semialdehyde dehydrogenase, part of the lysinebiosynthetic pathway).

Alternatively, the screening system may operate in an intact, livingmulticellular organism, such as an insect or a mammal. Methods ofgenerating transgenic Drosophila, mice and other organisms, bothtransiently and stably, are well known in the art; detection offluorescence resulting from the expression of Green Fluorescent Proteinin live Drosophila is well known in the art. One or more gene expressionconstructs encoding one or more of a labeled natural binding domain,sequence or polypeptide, a binding partner, a protein kinase orphosphatase and, optionally, a candidate modulator thereof areintroduced into the test organism by methods well known in the art (seealso below). Sufficient time is allowed to pass after administration ofthe nucleic acid molecule to allow for gene expression, for binding of anatural binding domain, sequence or polypeptide to its binding partnerand for chromophore maturation, if necessary (e.g., Green FluorescentProtein matures over a period of approximately 2 hours prior tofluorescence) before FRET is measured. A reaction component(particularly a candidate modulator of enzyme function) which is notadministered as a nucleic acid molecule may be delivered by a methodselected from those described below.

Dosage and administration of a labeled natural binding domain, sequenceor polypeptide, binding partner therefor, protein kinase or phosphataseor canditate modulator thereof for use in an in vivo assay of theinvention

Dosage

For example, the amount of each labeled natural binding domain orbinding partner therefor must fall within the detection limits of thefluorescence-measuring device employed. The amount of an enzmye orcandidate modulator thereof will typically be in the range of about 1μg-100 mg/kg body weight. Where the candidate modulator is a peptide orpolypeptide, it is typically administered in the range of about 100-500μg/ml per dose. A single dose of a candidate modulator, or multipledoses of such a substance, daily, weekly, or intermittently, iscontemplated according to the invention.

A candidate modulator is tested in a concentration range that dependsupon the molecular weight of the molecule and the type of assay. Forexample, for inhibition of protein/protein or protein/DNA complexformation or transcription initiation (depending upon the level at whichthe candidate modulator is thought or intended to modulate the activityof a protein kinase or phosphatase according to the invention), smallmolecules (as defined above) may be tested in a concentration range of 1pg-100 μg/ml, preferably at about 100 pg-10 ng/ml; large molecules,e.g., peptides, may be tested in the range of 10 ng-100 μμg/ml,preferably 100 ng-10 μg/ml.

Administration

Generally, nucleic acid molecules are administered in a mannercompatible with the dosage formulation, and in such amount as will beeffective. In the case of a recombinant nucleic acid encoding a naturalbinding domain and/or binding partner therefor, such an amount should besufficient to result in production of a detectable amount of the labeledprotein or peptide, as discussed above. In the case of a protein kinaseor phosphatase, the amount produced by expression of a nucleic acidmolecule should be sufficient to ensure that at least 10% of naturalbinding domains or binding partners therefor will undergo modificationif they comprise a target site recognized by the enzyme being assayed.Lastly, the amount of a nucleic acid encoding a candidate modulator of aprotein kinase or phosphatase of the invention must be sufficient toensure production of an amount of the candidate modulator which can, ifeffective, produce a change of at least 10% in the effect of the targetprotein kinase or phosphatase on FRET or other label emission resultingfrom binding of a natural binding domain to its binding partner or, ifadministered to a patient, an amount which is prophylactically and/ortherapeutically effective.

When the end product (e.g. an antisense RNA molecule or ribozyme) isadministered directly, the dosage to be administered is directlyproportional to the amount needed per cell and the number of cells to betransfected, with a correction factor for the efficiency of uptake ofthe molecules. In cases in which a gene must be expressed from thenucleic acid molecules, the strength of the associated transcriptionalregulatory sequences also must be considered in calculating the numberof nucleic acid molecules per target cell that will result in adequatelevels of the encoded product. Suitable dosage ranges are on the orderof, where a gene expression construct is administered, 0.5- to 1 μg, or10 μg, or optionally 10-100 μg of nucleic acid in a single dose. It isconceivable that dosages of up to lmg may be advantageously used. Notethat the number of molar equivalents per cell vary with the size of theconstruct, and that absolute amounts of DNA used should be adjustedaccordingly to ensure adequate gene copy number when large constructsare injected.

If no effect (e.g., of a protein kinase or phosphatase or an inhibitorthereof) is seen within four orders of magnitude in either direction ofthe starting dosage, it is likely that a protein kinase or phosphatasedoes not recognize the target site of the natural binding domain (and,optionally, its binding partner) according to the invention, or that thecandidate modulator thereof is not of use according to the invention. Itis critical to note that when high dosages are used, the concentrationmust be kept below harmful levels, which may be known if an enzyme orcandidate modulator is a drug that is approved for clinical use. Such adosage should be one (or, preferably, two or more) orders of magnitudebelow the LD₅₀ value that is known for a laboratory mammal, andpreferably below concentrations that are documented as producingserious, if non-lethal, side effects.

Components of screening assays of the invention may be formulated in aphysiologically acceptable diluent such as water, phosphate bufferedsaline, or saline, and further may include an adjuvant. Adjuvants suchas incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,or alum are materials well known in the art. Administration of labeledpolypeptides comprising a natural binding domain, sequence, polypeptideor a binding partner therefor, a protein kinase or phosphatase or acandidate modulator as described herein may be either localized orsystemic.

Localized administration:

Localized administration of a component of an assay of the invention ispreferably by via injection or by means of a drip device, drug pump ordrug-saturated solid matrix from which the labeled natural bindingdomain, sequence or polypeptide, binding partner therefor, proteinkinase or phosphatase or candidate modulator therefor, or nucleic acidencoding any of these can diffuse implanted at the target site. When atissue that is the target of delivery according to the invention is on asurface of an organism, topical administration of a pharmaceuticalcomposition is possible.

Compositions comprising a composition of- or of use in the inventionwhich are suitable for topical administration can take one of severalphysical forms, as summarized below:

(i) A liquid, such as a tincture or lotion, which may be applied bypouring, dropping or “painting” (i.e. spreading manually or with a brushor other applicator such as a spatula) or injection.

(ii) An ointment or cream, which may be spread either manually or with abrush or other applicator (e.g. a spatula), or may be extruded through anozzle or other small opening from a container such as a collapsibletube.

(iii) A dry powder, which may be shaken or sifted onto the target tissueor, alternatively, applied as a nebulized spray.

(iv) A liquid-based aerosol, which may be dispensed from a containerselected from the group that comprises pressure-driven spray bottles(such as are activated by squeezing), natural atomizers (or “pump-spray”bottles that work without a compressed propellant) or pressurizedcanisters.

(v) A carbowax or glycerin preparation, such as a suppository, which maybe used for rectal or vaginal administration of a therapeuticcomposition.

In a specialized instance, the tissue to which a candidate modulator ofa protein kinase or phosphatase is to be delivered for assay (or, iffound effective, for therapeutic use) is the lung. In such a case theroute of administration is via inhalation, either of a liquid aerosol orof a nebulized powder of. Drug delivery by inhalation, whether fortopical or systemic distribution, is well known in the art for thetreatment of asthma, bronchitis and anaphylaxis. In particular, it hasbeen demonstrated that it is possible to deliver a protein via aerosolinhalation such that it retains its native activity in vivo (see Hubbardet al., 1989, J. Clin. Invest., 84: 1349-1354).

Systemic administration:

Systemic administration of a protein, nucleic acid or other agentaccording to the invention may be performed by methods of whole-bodydrug delivery are well known in the art. These include, but are notlimited to, intravenous drip or injection, subcutaneous, intramuscular,intraperitoneal, intracranial and spinal injection, ingestion via theoral route, inhalation, trans-epithelial diffusion (such as via adrug-impregnated, adhesive patch) or by the use of an implantable,time-release drug delivery device, which may comprise a reservoir ofexogenously-produced protein, nucleic acid or other material or may,instead, comprise cells that produce and secrete a natural bindingdomain and/or a binding partner therefor, protein kinase or phosphataseor candidate modulator thereof. Note that injection may be performedeither by conventional means (i.e. using a hypodermic needle) or byhypospray (see Clarke and Woodland, 1975, Rheumatol. Rehabil., 14:47-49). Components of assays of the invention can be given in a single-or multiple dose.

Delivery of a nucleic acid may be performed using a delivery techniqueselected from the group that includes, but is not limited to, the use ofviral vectors and non-viral vectors, such as episomal vectors,artificial chromosomes, liposomes, cationic peptides, tissue-specificcell transfection and transplantation, administration of genes ingeneral vectors with tissue-specific promoters, etc.

Kits According to the Invention

A kit for assaying the activity of a protein kinase or phoshatase

In order to facilitate convenient and widespread use of the invention, akit is provided which contains the essential components for screeningthe activity of a protein kinase or phosphatase, as described above. Anatural binding domain, sequence or polypeptide, as defined above, andits corresponding binding partner are provided, as is a suitablereaction buffer for in vitro assay or, alternatively, cells or a celllysate. A reaction buffer which is “suitable” is one which is permissiveof the activity of the enzyme to be assayed and which permitsphosphorylation-dependent binding of the natural binding domain to thebinding partner. The labeled polypeptide components are provided aspeptide/protein or a nucleic acid comprising a gene expression constructencoding the one or more of a peptide/protein, as discussed above.Natural binding domains, sequences and polypeptides, as well as theircorresponding binding partners, are supplied in a kit of the inventioneither in solution (preferably refrigerated or frozen) in a buffer whichinhibits degradation and maintains biological activity, or are providedin dried form, i.e., lyophilized. In the latter case, the components areresuspended prior to use in the reaction buffer or other biocompatiblesolution (e.g. water, containing one or more of physiological salts, aweak buffer, such as phosphate or Tris, and a stabilizing substance suchas glycerol, sucrose or polyethylene glycol); in the latter case, theresuspension buffer should not inhibit phosphorylation-dependent bindingof the natural binding domain, sequence or polypeptide with the bindingpartner when added to the reaction buffer in an amount necessary todeliver sufficient protein for an assay reaction. Natural bindingdomains, sequences or polypeptides or their binding partners provided asnucleic acids are supplied- or resuspended in a buffer which permitseither transfection/transformation into a cell or organism or in vitrotranscription/translation, as described above. Each of these componentsis supplied separately contained or in admixture with one or more of theothers in a container selected from the group that includes, but is notlimited to, a tube, vial, syringe or bottle.

Optionally, the kit includes cells. Eukaryotic or prokaryotic cells, asdescribed above, are supplied in- or on a liquid or solid physiologicalbuffer or culture medium (e.g. in suspension, in a stab culture or on aculture plate, e.g. a Petri dish). For ease of shipping, the cells aretypically refrigerated, frozen or lyophilized in a bottle, tube or vial.Methods of cell preservation are widely known in the art; suitablebuffers and media are widely known in the art, and are obtained fromcommerical suppliers (e.g., Gibco/LifeTechnologies) or made by standardmethods (see, for example Sambrook et al., 1989, Molecular Cloning. ALaboratory Manual., 2nd Eddition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

An enzyme being assayed according to the invention is added to the assaysystem either as a protein (isolated, partially-purified or present in acrude preparation such as a cell extract or even a living cell) or arecombinant nucleic acid. Methods of expressing a nucleic acidcomprising an enzyme or other protein are well known in the art (seeagain above).

An assay of the invention is carried out using the kit according to themethods described above and in the Examples.

A kit for screening a candidate modulator of protein kinase orphosphatase activity according to the invention

A candidate modulator of post-translational phosphorylation ordephosphorylation may be assayed using a kit of the invention. A kit asdescribed above is used for this application, with the assay performedfurther comprising the addition of a candidate modulator of the proteinkinase or phosphatase which is present to the reaction system.Optionally, a protein kinase or phosphatase is supplied with the kit,either as a protein or nucleic acid as described above.

Assays of protein activity are performed as described above. At aminimum, three detections are performed, one in which the naturalbinding domain and binding partner are present without the proteinkinase or phosphatase or candidate modulator thereof (control reactionA), one in which the polypeptides are incubated with the modifyingenzyme under conditions which permit the phosphorylation ordephosphorylation reaction to occur (control reaction B) and one inwhich the protein kinase or phosphatase and candidate modulator are bothincubated with the labeled polypeptides under conditions which permitthe modification reaction to occur (test reaction). In each case,conditions are suitable to permit phosphorylation-dependent associationof the natural binding domain, sequence or polypeptide and the bindingpartner. The result of the last detection procedure is compared withthose of the two controls; the candidate modulator is judged to beefficacious if there is a shift in either of the observed amount ofsignal (i.e., total amount- or rate of change of fluorescence, FRET,mass of a protein complex or inhibition or activation of an enzyme) ofat least 10% away from that observed in control reaction B toward thatobserved in control reaction A.

EXAMPLE 1

Use of a polypeptide comprising a natural binding domain as aphosphorylation reporter according to the invention: Assay 1

An assay of this type involves the following components:

v-Src SH2 domain (amino acids 148-246; Waksman et al., 1993, Cell, 72:779-790; OWL database accession no. M33292), and

Hamster polyomavirus middle T antigen (Ag, below) (321-331, EPQYEEIPIYL,SEQ ID NO: 4; Waksman et al., 1993, supra; OWL database accession no.P03079).

SH2 domains are found in proteins involved in a number of signallingpathways and their binding to specific phosphorylated tyrosine residuesis key in mediating the transmission of signals between tyrosine kinasesand the proteins in the cell which respond to tyrosine phosphorylation(Waksman et al., 1993, supra and references therein). Individual SH2domains recognize specific sequences, and the sequence specificity of anumber of SH2 domains has been determined (Songyang et al., 1993, supra)using a phosphopeptide library. These data provide a number of possibledomain/peptide pairs which are useful in assays of enzymatic activityaccording to the invention. The crystal structure of the Src SH2 domaincomplexed with a peptide containing its specific recognition motif fromthe hamster middle-T antigen (target tyrosine for phosphorylation shownin bold above) has been determined by Waksman et. al.(Cell 72, 779-790).Thus, the assay is:

SH2-F1 + Ag-F2 ⇄ (SH2-F1)(PAg-F2) No FRET FRET

F1 is the donor fluorophore, F2 the acceptor fluorophore and P denotesthe addition of a phosphate group to the target tyrosine residue.

The peptide as used in the crystallization described above does notcontain suitable residues for convenient labelling, and a label withinthis short sequence is too close to the phosphorylation site. A shortlinker (e.g., Gly-Gly) is, therefore, added to either the C- orN-terminus of the peptide with a residue such as Lys for labelling onthe end. The location of this linker will depend upon the location of F1in the SH2 domain.

A number of potential locations for the fluorophore in the SH2 domainhave been identified based upon crystal structure:

SH2 Domain Middle T Ag. peptide K 232 C-terminal extension (G-G-K orsimilar) R 217 C-terminal extension K 181 N-terminal extension R 156**N-terminal extension **this site is close to the site of peptideinteraction

If a fluorescent protein (e.g., Green Fluorescent Protein, GFP) is usedinstead of a chemical fluorophore, it is placed at the N-termini of boththe SH2 domain and the peptide

EXAMPLE 2

Use of apolypeptide comprising a natural bindmg domain as aphosphorylation reporter according to the invention: Assay 2

This assay involves the following components:

PTB domain of IRS-1 (amino acids 157-267) (Zhou et al., 1996, NatureStuctural Biology, 3: 388-393; OWL accession no. P35568), and

Interleukin 4 Receptor (IL-4R) (amino acids 489-499, LVIAGNPAYRS, SEQ IDNO:5; Zhou et. al., 1996, supra; OWL accession no. P24394)Phosphotyrosine binding (PTB) domains are found in a number of proteinsinvolved in signalling pathways utilizing tyrosine phosphorylation. ThePTB domain has functional similarities to the SH2 domain but differs inits mechanism of action and structure, as well as in sequencerecognition (Laminet et al., 1996, J. Biol, Chem., 271: 264-269; Zhouet. al., 1996, supra and references therein). These two classes ofdomain have little sequence identity. NMR structural analysis of the PTBdomain of IRS- 1 complexed with the IL-4 receptor peptide has beenperformed (Zhou et al., 1996, supra).

The assay format is as follows:

PTB-F1 + IL-4R-F2 ⇄ (PTB-F1)(PIL-4R-F2) No FRET FRET

F1 is the donor fluorophore, F2 the acceptor fluorophore, and P denotesthe addition of a phosphate group to the target tyrosine residue.

The peptide of the NMR study described above does not contain suitableresidues for convenient labelling except the arginine next to thephosphorylation site, and a label within this short sequence may be tooclose to the target site for phosphorylation. A short linker may beadded to either the C- or N-terminus of the peptide with a residue forlabelling on the end. The location of such a linker depends upon thelocation of F1 in the PTB domain.

Several potential locations for the fluorophore in the PTB sequence havebeen identified from the NMR structure:

PTB domain IL-4R peptide K161 N-terminal extension (G-G-K or similar)K190 N-terminal extension N-terminal extension N-terminal extensionC-terminal extension C-terminal extension

Again, if GFP is used in lieu of a chemical fluorophore, it can be fusedin-frame to either the N- or C-terminus of both the PTB sequence and thebinding partner.

EXAMPLE 3

An assay analogous to that in Example 2 can be configured according tothe invention using the PTB domain of the proto-oncogene product Cbl anda peptide derived from the Zap-70 tyrosine kinase. The Cblphosphotyrosine-binding domain selects a D(N/D)XpY motif and binds tothe Tyr₂₉₂ negative regulatory phosphorylation site of ZAP-70 (Lupher etal., 1997, J. Biol. Chem., 272: 33140-33144).

The components of the assay are:

The Cbl N-terminal domain (amino acids 1-357; Lupher et al., 1996, J.Biol. Chem., 271: 24063-24068; OWL accession no. P22681), and

Zap-70 (amino acids 284-299, NH₃-IDTLNSDGYTPEPARI-COOH, SEQ ID NO:6;Lupher et. al., 1996, supra; OWL accession no. P43403)

EXAMPLE 4

Use of a polypeptide comprising a natural binding domain as aphosphorylation reporter according to the invention: Assay 4

This assay involves the following component-c-Src (residues 86-536; Xuet al., 1997, Nature, 385: 595-602; GenBank Accession No. K03218).

As stated above, Src is a member of a family of non-receptor tyrosinekinases involved in the regulation of responses to extracellularsignals. Association of src with both the plasma membrane andintracellular membranes is mediated by myristoylation at the N-terminus.The enzyme has four regions which are conserved throughout the family,the SH2 domain, the SH3 domain, the kinase or SH1 domain and theC-terminal tail. In addition there is a unique region which does nothave homology between family members (Brown and Cooper, 1996,BiochimBiophysActa, 1287: 121-149).

The SH2 domain binds tightly to specific tyrosine phosphorylatedsequences. This affinity plays a role in the interaction between src andother cellular proteins and also in the regulation of the kinase byphosphorylation. The C-terminal tail of src can be s phosphorylated onTyr,₃₀, which phosphorylation leads to almost complete inhibition ofkinase activity. There is strong evidence that this inhibition isachieved by the interaction of the C-terminal tail with the SH2 domain.This interaction is thought to promote a conformational change to the‘closed’ conformation which is further stabilized by the participationof the SH3 and kinase domains in intramolecular contacts.

The assay is diagramed as follows:

Src-SH2-F1--------Tail-F2 + ATP ⇄ Src-(SH2-F1)(PTail-F2) + ADP No FRETFRET

where F1 is the donor fluorophore, F2 is the acceptor fluorophore and Pdenotes the addition of a phosphate group to the target tyrosineresidue.

There are several potential sites for labelling in this structure. Someexamples of target residues are shown below:

C-Terminal tail SH2 domain E527 D195, K198 C-Terminal extension (eg.Gly-Gly-Lys) R220, K235,

When a fluorescent protein is used in an assay such as this, using anintramolecular interaction to follow chemical modification, it isappropriate to place GFP between domains using a flexible linker topreserve protein domain interactions. This allows the GFP variants toapproach more closely and increase the efficiency of the FRET achieved,but must be balanced by the need to achieve a good distance betweenvariants in the ‘No FRET’ state. If sufficient spacing between donor andacceptor fluorophores or, alternatively, between a fluorophore or otherlabel and a quencher therefor, is not achieved in this manner, othercandidate locations for fluorescent protein fusion include, but are notlimited to, the C-terminus and the region between the SH2 domain and theSH2-kinase linker.

EXAMPLE 5

Solution FRET assay for Yersinia Tyrosine Phosphatase (YOP) using anatural binding partner labeled with a fluroescent protein and asynthetic peptide labelled with a chemical fluorophore

The following solution based assay was performed to detect YOP activityby measuring disruption of a complex between a fluorescently labelledSH2 domain of ZAP-70 and a synthetic peptide based on the TCRC chainlabelled with a chemical fluorophore.

A FRET partnership is formed between the SH2 domain of ZAP-70 and aphosphorylated peptide based on the TCRζ chain, providing both partnersare labelled with suitable fluorophores. Formation of FRET is followedin real-time by adding the two binding partners together. Disruption ofFRET can be achieved by the addition of a phosphatase, which removes thephosphate required for the interaction of the partners.

Methods: TCR peptide sequence Peptide 1. Phosphorylated TCRζ chain:RCKFSRSAEPPAYQQGQNQLY_((p))NELNLGRREEY_((p))DVLD, SEQ ID NO:7

Peptide labelling

Peptide 1 was labelled with rhodamine under mild conditions using thioldirected chemistry. 230 μM peptide was labelled in 2 mM TES pH 7 in thepresence of a three-fold excess of rhodamine-6-maleimide (MolecularProbes). Dialysis was utilised to remove excess dye from the peptide.Labelling was verified by MALDI-TOF MS.

ZAP-GP coningand purification

DNA constructs

ZAP-GFP: Primers were designed based on the published ZAP-70 DNAsequence (Genbank accession number L05148). The SH2 domain (amino acids1-259) of ZAP70 was cloned by PCR using the following oligo-nucleotides:

Forward primer GGGATCCATATGCCAGACCCCGCGGCGCACCTG, SEQ ID NO:8

Reverse Primer GGAATTCGGGCACTGCTGTTGGGGCAGGCCTCC, SEQ ID NO:9

The resultant PCR fragment was digested with BamHI and EcoRI andinserted into pET28a (Novagen) to generate vector pFS45. DNA encodingGFP in the vector pQBI25-FNI (Quantum) was digested with MluI and theresultant 5′ overhang was “filled in” using T4 DNA polymerase (NEB).After the polymerase was denatured by heat treatment the DNA was furtherdigested with EcoRI and the resultant 850 bp band was gel purified. Thevector pFS45 was digested with Hindlll and the resultant 5′ overhang was“filled in” with T4 DNA polymerase and then further digested with EcoRI.After the digested vector was gel purified it was ligated with thepurified DNA encoding GFP to generate pFS46 which was designed toexpress a ZAP70-GFP fusion protein in bacteria.

Expression and purification procedure

Fresh transformants of ZAP-GFP pET-28a in BRL(DE3) were used toinoculate 3 ml LB/kanamycin (100 μ/ml). The starter cultures wereincubated overnight at 37° C. with constant shaking. From these startercultures Iml was used to inoculate 400 ml Terrific Broth/kanamycin (100μg/ml) in a 2L, baffled flask. Cultures were incubated at 37° C. at 2rpm for approximately 5 hrs until the OD600 nm reached 0.5Abs units. Atthis point cultures were induced by adding IPTG to a concentration oflmM. The cultures were then left incubating at room temperatureovernight with gentle shaking on a benchtop rotator. Bacteria wereharvested by centrifugation at 3000 rpm for 20 mins. The bacterialpellet was resuspended in 25 ml lysis buffer (5 mM phosphate buffer pH7.0, 300 mM NaCl, 2% Proteinase inhibitor cocktail (Sigma), 0.75 mg/mlLysozyme). Lysis of the resuspended cells was initiated by gentlestirring for lhr at room temperature. The partially lysed mixture wassubjected to 2 cycles of freeze thawing in liquid nitrogen. Finally thecells were sonicated on ice using a 10 mm probe at high power.Sonication was performed on a pulse setting for a period of 3 min. Thecrude lysate was then centrifuged at 15,000 rpm for 30 mins to removecell debris. Hexa-His tagged proteins were purified from the clearlysate using TALON® resin (Clontech). Proteins were bound to the resinin a batchwise manner by gentle shaking at room temperature for 30 min.Non-His tagged proteins were removed by washing the resin at least twicewith 10× bed volume of wash buffer (50 mM sodium phosphate pH 7.0, 3 mMNaCl, 5 mM fluorescence-blank Imidazole ). The washed resin was loadedinto a 2 ml column and the bound proteins were released with elutionbuffer (50 mM sodium phosphate pH 7.0, 300 mM NaCl, 150 mMfluorescence-blank Imidazole). Elution was normally achieved after thefirst 0.5 ml and within 2-3 ml in total. Proteins were stored at −80° Cafter snap freezing in liquid nitrogen in the presence of 10% glycerol.

Formation of the FRET partnership

ZAP-GFP was diluted to 0.5 μM in YOP assay buffer (5 mM Tris-HCI pH 7.2,10 mM β-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35). 98 μl of thissolution was used per assay. Initial readings of the fluorescence of theZAP-GFP construct were made using 485nm excitation wavelength and 520nmemission wavelength. Rhodamine labelled peptide (2jl of a 115 μM peptidesolution) was added to the ZAP-GFP and formation of FRET followed inreal-time by measuring the decrease in fluorescence emission of ZAP-GFPat 520 nm.

YOP source and assay conditions

Yersinia protein tyrosine phosphatase (YOP) was purchased from UpstateBiotechnology. YOP, 3 units, was added to the ZAP-GFP/peptide FRETmixture and the increase in fluorescence emission at 520 nm, wasfollowed in real-time as the partnership was disrupted (FIG. 4).Dependence of YOP activity on TCR peptide concentration and Kmdetermination was measured using different concentrations of rhodaminelabelled TCRζ peptide (FIG. 5).

The solution FRET assay for YOP was also used to determine the IC₅₀ ofortho vanadate, a general protein phosphatase inhibitor. The YOP assaywas performed as above, using 3 units of YOP and incubating the enzymewith the ZAP-GFP/peptide FRET mixture at 30° C. Dephosphorylation wasfollowed in real-time by measuring the increase in fluorescence at 520nm. Sodium orthovanadate was added to the reaction mix prior to enzymeaddivity to give final concentrations of (0.03-30 μM). Results indicatean IC₅₀ value for vanadate of 0.25 μM and are shown in FIG. 6.

EXAMPLE 6 Assay of Chk1 Kinase Using Solution Phase FP Assay withFluorescein Labelled CDC25 Derived Peptide Substrate and 14-3-3ζ BindingPartner

This assay was performed to measure Chk1 activity by measuring thefluorescence polarisation change that occurs as a result of the Chk1protein kinase-mediated binding of fluorescein labelled Chktide to14-3-3 protein.

Chk1 protein kinase modifies the activity of CDC25 phosphatase viaserine phosphorylation. Phosphorylation of CDC25 results in the bindingof different isoforms of the 14-3-3 protein and subsequent inhibition ofCDC25 phosphatase activity. A peptide derived from CDC25 (Chktide) andlabelled with a chemical fluorophore binds to 14-3-3 isoforms zeta andepsilon when phosphorylated by Chk1 kinase. The activity of Chk1 ismonitored by following the fluorescence polarisation change whenfluorescein labelled Chktide binds to 14-3-3 protein.

Methods

Reagent sources

14-3-3-ζ protein, Chk1 enzyme and Ckhtide peptide are from UpstateBiotechnology. Chktide, Chk1 substrate peptide sequence:KKKVSRSGLYRSPS²¹⁶MPENLNRPR, SEQ ID NO:10

Chktide labelling with fluorescein

Chktide was labelled by incubating the peptide for 2 hours at aconcentration of 0.185 mM in 100 mM NaHCO₃, pH 8.3, and 0.37 mMfluorescein-5EX (Molecular Probes) at room temperature. The labelledpeptide was then dialysed against 3 changes of 50 mM Tris HCL pH 7.4,150 mM NaCl (200 ml) for a total of 18 hours.

Phosphorylation of Chktide by Chk1 protein kinase.

A peptide substrate for Chk1 (Chktide) was labelled with fluorescein.The labelled peptides were then phosphorylated by incubating them at aconcentration of 30 μM for 30 min at 30° C. in 20 mM MOPS pH 7.2, 10 mMMgCl₂, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1mM DTT, 0.1 mM ATP, and 62 mU Chk1. Non phosphorylated control sampleswere prepared by incubation of the labelled peptides under identicalconditions in the absence of Chk1. The peptides were then incubated at aconcentration of 5 μM in 20 mM MOPS pH 7.2 at 30° C., in a total volumeof 30 μl on a half area 96 well plate. The fluorescence polarisation ofthe samples was measured at 520 nm (excitation 485 nm) following a 5 minequilibration. 14-3-3ζ (5 μM) was added to the peptide samples, and thefluorescence polarisation at 520nm (excitation 485 nm) was monitoredover time as shown in FIG. 7. Inhibition of Chk1 activity by EDTA wasmeasured by following the above procedure and adding EDTA (10 mM or 20mM) prior to enzyme addition, as shown in FIG. 8.

Activity of Chk1 was monitored in real time as follows. Fluoresceinlabelled Chktide was incubated at 30° C. in a total volume of 30 μl (ona half area 96 well plate) in 20 mM MOPS pH 7.2, 10 mM MgCl₂, 25 mMβ-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, and0.1 mM ATP in the presence of 5 μM 14-3-3 ζ. The samples were allowed toequilibrate for 5 min, then Chk1 was added (or an equal volume of H₂Ofor control samples) and the fluorescence polarisation at 520 nm(excitation 485 nm) was monitored over time as shown in FIG. 9.Dependence of the increased fluorescence polarisation signal on Chk1activity and 14-3-3ζ binding was shown by the lack of binding when ATPor 14-3-3ζ protein was omitted from the real time enzyme reaction asshown in FIG. 10. Inhibition of Chk1 activity by EDTA was measured byfollowing the above procedure and adding EDTA (1 mM, 5 mM or 20 mM)prior to the addition of 21 mU of Chk1 to start the reaction, as shownin FIG. 11. The fluorescence polarisation for each sample was determinedat the end of the linear portion of the reaction.

Chk1 activity was also measured by monitoring the binding ofphosphorylated Chktide peptide to an alternate isoform of the 14-3-3protein, 14-3-3ε.

Production of 14-3-3 ε

Under the control of the T7 promoter, the vector FS 121 contains DNAencoding the 14-3-3ε (Genbank accession number U54778) protein fusedin-frame to DNA encoding an amino terminal hexa-His tag. Freshtransformants of pFS 121 in BRL(DE3) pLysS were used to inoculate 3 mlLB/ampicillin (100 μg/ml). The starter cultures was incubated overnightat 37° C. with shaking. From these starter cultures 1 ml was used toinoculate 400 ml Terrific Broth/ampicillin (100 μg/ml) in a 2L, baffledflask. Cultures were incubated at 37° C. at 200 rpm for approximately 4hr until the OD₆₀₀ nm reached 0.5 Abs units. At this point cultures wereinduced by adding IPTG to a concentration of ImM and further incubatedat 37° C. for 4 hrs.

Bacteria were harvested by centrifugation at 3000 rpm for 20 min. Thebacterial pellet was resuspended in 25 ml lysis buffer (50 mM PhosphatepH 7.0, 30 mM NaCl, 2% Proteinase inhibitor cocktail (Sigma), 0.75 mg/mlLysozyme). Lysis of the resuspended cells was initiated by gentlestirring for 30 min at room temperature. Nonidet P-40 was added to afinal concentration of 1% and lysis was continued for an additional 20min at room temperature. The partially lysed mixture was subjected to 3cycles of freeze thawing in liquid nitrogen. Finally the cells weresonicated on ice using a 10 mm probe at high power. Sonication wasperformed on a pulse setting for a period of 4 min. The crude lysate wascentrifuged at 15,000 rpm for 30 min to remove cell debris. Hexa-Histagged proteins were purified from the cleared lysate using TALON® resin(Clontech). Proteins were bound to the resin in a batchwise manner bygentle shaking at room temperature for 30 min. Non-His tagged proteinswere removed by washing the resin at least twice with a 10× bed volumeof wash buffer (50 mM sodium phosphate pH 7.0, 300 mM NaCl, 5 mMfluorescence-blank Imidazole ). The washed resin was loaded into a 2 mlcolumn and the bound proteins were released with elution buffer (50 mMsodium phosphate, pH 7.0, 300 mM NaCl, 150 mM fluorescence-blankImidazole). Elution was normally achieved within 5 ml. Purified proteinswere stored at −80° C. after snap freezing in liquid nitrogen in thepresence of 10% glycerol.

End point assay of Chk1 kinase by 14-3-3ε binding to phosphorylatedChktide

A peptide substrate for Chk1 (Chktide) was labelled with fluorescein.The labelled peptides were then phosphorylated by incubating them at aconcentration of 30 μM for 30 min at 30° C. in 100 l of 20 mM MOPS pH7.2, 10 mM MgCl₂, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodiumorthovanadate, 1 mM DTT, 0.1 mM ATP, and 62 mU Chk1. Non-phosphorylatedcontrol samples were prepared by incubation of the labelled peptidesunder identical conditions in the absence of Chk1. The peptides werethen incubated at a concentration of 7.5 μμM in 20 mM MOPS pH 7.2 at 30°C., in a total volume of 30 μl on a half area 96 well plate. Thefluorescence polarisation of the samples was measured at 520 nm(excitation 485 nm) following a Smin equilibration. 14-3-3 ε (4 μl) wasadded to the peptide samples, and the fluorescence polarisation at 520nm (excitation 485 nm) was monitored over time as shown in FIG. 12.

EXAMPLE 7 Solution phase FP assay for the detection of phosphatase λactivity using a fluorescein labelled CDC25 derived peptide substrateand 14-3-3ζ binding partner

These assays were performed to demonstrate measurement of phosphatase 1activity as detected by a change in fluorescence polarisation due todecreased binding of Chktide to 14-3-3 ζ or 14-3-3 ε. Binding isdecreased as a result of dephosphorylation of Chktide by phosphatase λ.

Reagents were obtained and prepared as in example 6 above.

Fluorescein labelled Chktide was phosphorylated by incubation at aconcentration of 30 μM for 30 min at 30° C. in 20 mM MOPS pH 7.2, 10 mMMgCl₂, 25 mM ε-glycerophosphate, 5 mM EGTA, 1 mMsodium orthovanadate, 1mM DTT, 0.1 mM ATP, and 62mU Chk1. Non-phosphorylated control sampleswere prepared by incubation of the labelled peptides under identicalconditions in the absence of Chk1. The peptides were then incubated at aconcentration of 7.5 μM in 20 mM MOPS pH 72, 2 mM Mncl₂, 1 mM DTT at 30°C., in a total volume of 30 μl on a half area 96 well plate. Thefluorescence polarisation of the samples was measured at 520 nm(excitation 485 nm) following a 5 min equilibration. 14-3-3 ζ (5 μM) wasadded to the peptide samples, and the fluorescence polarisation at 520nm (excitation 485 nm) was monitored over time as shown in FIG. 13.Alternatively 14-3-3 ε (4 μl of purified protein prepared as in Example6) was added to the peptide samples, and after the fluorescencepolarisation was stabilised, phosphatase λ(200U) was added. Thefluorescence polarisation at 520 nm (excitation 485 nm) was monitoredover time as shown in FIG. 14.

EXAMPLE 8 Simultaneous Assay of Two Serine Threonine Kinases, Chk1 andcAMP Dependent Protein Kinase (PKA) by FP

This assay was performed to simultaneously measure the activity of Chk1and PKA by monitoring a change in fluorescence polarisation due toassociation or dissociation of peptides and/or proteins capable ofassociating in a manner that is dependent upon their phosphorylationstate. Following phorphorylation by PKA, coiled coil dimers can nolonger form. Conversely, phosphorylation of Chktide by Chk1 results inbinding of Chktide to a 14-3-3 protein.

Serine threonine kinases PKA and Chk1 can be assayed simultaneouslyusing the natural binding partner reporters described in example 5 forChk1 and a peptide based binding partner assay for PKA (described inWO99/11774).

Methods

PKA Peptide sequences:

Peptide 1. ERE IKALERE IRRLRRA SQALERE IAQLERE, SEQ ID NO:11

Peptide 2. LRQR IQCLRYR IRRLRRA SQALRQR IAQLKQR, SEQ ID NO:12

PKA peptides form coiled-coil dimers when they are non-phosphorylated.Each monomer has a PKA phosphorylation site. Following phosphorylationby PKA, the dimers can no longer form. The association/dissociation canbe measured using a fluroescence polarisation assay, where PKA peptide 1is labelled with coumarin, and peptide 2 is labelled with biotin andbound to streptavidin. The tumbling rate of coumarin labelled peptide 1is higher after PKA phosphorlation and dissociation from thedimer/streptavidin complex. At the same time, the activity of Chk1 ismeasured by the decrease in tumbling rate of phosphorylated fluoresceinlabelled Chktide substrate binding to 14-3-3ε protein.

The labelled PKA peptides (both at a concentration of 2.5 μM) wereincubated at 30° C. in a total volume of 50 μ (on a half area 96 wellplate) in 20 mM MOPS pH 7.2, 10 mM MgCl₂, 25 mM β-glycerophosphate, 5 mMEGTA, 1 mM sodium orthovanadate, 1 mM DTT, and O.ImM ATP, 7.5 μMChktide, 3 μL 14-3-3 ε and 0.06U streptavidin. The samples were allowedto equilibrate for 5 min, then CHK1 (62 mU) and PKA (0.5 pmoles) wereadded (or an equal volume of H₂O for control samples). The fluorescencepolarisation at 520 nm (excitation 485 nm) and at 450 nm (excitation 340nm) was monitored over time as shown in FIG. 15.

EXAMPLE 9 FRET Assay of Src Using ZAP70-GFP Binding Partner andSynthetic Rhodamine Labelled TCRζ Substrate

These FRET-based assays were performed to detect Src activity bymeasuring the Src dependent formation of a complex between an SH2 domainof ZAP-70 labelled with a fluorophore and a peptide based on the TCRζchain labelled with a fluorophore.

A FRET partnership can be formed in a phosphorylation dependent mannerbetween the SH2 domain of ZAP-70 and a peptide based on the TCRζ chain,providing both binding partners are labelled with suitable fluorophores.Src is used to phosphorylate the TCRζ chain derived substrate.

Methods:

TCR peptide sequence

Peptide 1. Phosphorylated TCRC chain:RCKFSRSAEPPAYQQGQNQLY_((p))NELNLGRREEY_((p))DVLD, SEQ ID NO: 13

Peptide 2. Unphosphorylated TCRζ chain:RCKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLD, SEQ ID NO: 14

Peptide labelling

Peptides 1 and 2 were labelled with rhodamine under mild conditionsusing thiol directed chemistry. 230 μM peptide was labelled in 2 mM TESpH 7 in the presence of a three-fold excess of rhodamine-6-maleimide(Molecular Probes). Dialysis was utilised to remove excess dye from thepeptide. Labelling was verified by MALDI-TOF MS.

ZAP-GEP cloning and purification

ZAP-GFP was cloned and purified as described in Example 5.

Phosphorylation of Peptide 2

Peptide 2 labelled with rhodamine was phosphorylated using 3 units ofSrc kinase (Upstate Biotechnology) in a 40 μl reaction containing 115 μMpeptide, 50 mM Tris-HCl pH 7.2, 1 mM ATP, 10 mM MgCl_(2, 10) mMβ-mercaptoethanbl, 0.1 mg/ml BSA and 0.015%(v/v) Brij 35, over a periodof 2 hours at 37° C. Non-phosphorylated control samples were preparedunder identical conditions in the absence of the kinase.

Formation of the FRET partnership

ZAP-GFP was diluted to 0.5 μM in assay buffer (50 mM Tris pH 7.2, 10 mMβ-mercaptoethanol, 0.5mg/ml BSA, 0.015% Brij 35). 98 μl of this solutionwas used per assay. Initial readings of the fluorescence of the ZAP-GFPconstruct were made using 485 nm excitation wavelength and 520 nmemission wavelength. Rhodamine labelled peptide (2 μl of abovephosphorylation reactions) was added to the ZAP-GFP solution andformation of FRET was followed in real-time by measuring the decrease influorescence emission of ZAP-GFP at 520 nm as shown in FIG. 16.

EXAMPLE 10 Solution Phase FRET Assay of Src Kinase Activity Using aNatural Binding Partner, SHP-2 Labelled with a Fluorescent Protein

These FRET-based assays are performed to measure Src kinase activity asdetermined by the Src kinase dependent formation of a complex betweenthe SH2 domain of SHP2 labelled with a fluorophore and an syntheticpeptide based on SHPS-1, also labelled with a fluorophore.

Interaction between the tandem SH2 domain of SHP2 and a syntheticpeptide based on SHPS-1 is mediated by the state of phosphorylation ofthe peptide. A FRET partnership can be established betweenphosphorylated peptide and the SH2 domain providing both moieties arelabelled with suitable fluorophores which are brought into closeproximity upon interaction of binding partners.

SUP2 peptide sequence and fluorescent labelling

SHPS-1 peptide sequence. BiotinKQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSC,SEQ ID NO:14

The SHPS-I was labelled using thiol directed chemistry. 200 μM peptidewas reacted with 600 μM rhodamine-6-maleimide (Molecular Probes) in 20mM TES pH 7 over a period of at least two hours at room temperature.Excess label was removed using dialysis and the labelling was verifiedby MALDI-TOF MS.

SHP2-GFP cloning and purification

Primers were based on the published SHP-2 DNA sequence (Genbankaccession number L03535. The SH2 domain (amino acids 1-225) of SHP-2 wascloned by PCR using the following oligo-nucleotides:

5′primer-GGGGATCCTCTAGAATGACATCGCGGAGATGGTTTCACCC, SEQ ID NO: 15

3′primer-GGGGAATTCTTTCAGCAGCATTTATACGAGTCG, SEQ ID NO:16

The resultant PCR fragment was digested with XbaI and EcoRI, gelpurified and ligated into pET28a (Novagen) to generate vector pFS114.The validity of the construct was confirmed by sequence analysis. DNAencoding GFP in the vector pFS46 was isolated by digestion with therestriction enzymes EcoRI and XhoI and the resultant 860 bp band was gelpurified and ligated into pFS114 to generate a bacterial expressionvector for production of the fusion protein SHP2-GFP (pFS115).

The hexa-His tagged SHP2-GFP fusion protein was expressed and purifiedusing TALON® resin according to standard procedures.

Phosphorylation of peptide SHPS-1 peptide

SHPS-1 peptide labelled with rhodamine was phosphorylated using 3 unitsof Src kinase (Upstate Biotechnology) in a 40 μl reaction containing 100μM peptide, 50 mM Tris-HCl pH 7.2, 1 mM ATP, 10 mM MgCl₂, 10 mMβ-mercaptoethanol, 0.1 mg/ml BSA and 0.015% (v/v) Brij 35, over a periodof 2 hours at 37° C. Non-phosphorylated control samples were preparedunder identical conditions in the absence of the kinase.

Formation of the FRET partnership

SHP2-GFP was diluted to 0.5 μM in assay buffer (50 mM Tris HCL pH 7.2,10 mM β-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35). 98 μl ofthissolution was used per assay. Initial readings of the fluorescence of theSHP2-GFP construct were made using 485 nm excitation wavelength and 520nm emission wavelength. Rhodamine labelled peptide (2 μl of the abovephosphorylation reactions) was added to the SHP2-GFP solution andformation of FRET was monitored in real-time by measuring the decreasein fluorescence emission of SHP2-GFP at 520 nm as shown in FIG. 17.

Disruption of the FRET partnership

The FRET partnership described in the above section was disrupted easilyusing the tyrosine phosphatase enzyme, YOP (Upstate Biotechnology). FRETpartnership was formed as in the above example, using 4 μl ofphosphorylated or control non-phosphorylated SHPS-1 peptide. Addition of3 units of YOP to the FRET partnership resulted in disruption of theFRET partnership as the phosphotyrosines required for the formation ofthe partnership were removed (shown in FIG. 18).

Inhibition by Staurosporine

Phosphorylation of the peptide was prevented by inhibiting the enzyme,Src, with the potent kinase inhibitor staurosporine. Phosphorylation ofthe rhodamine labelled SHPS-1 peptide was performed as described abovein the presence or absence of 10 μM staurosporine added to the reactionprior to the addition of the Src enzyme. The SH2 domain of SHP2 and theSHPS-1 peptide failed to form a FRET partnership in the presence of theinhibitor, as shown in FIG. 19.

Assay of Src phosphorylation of SHPS-1 peptide in real-time

Phosphorylation of the rhodamine labelled SHPS-1 peptide and formationof the FRET partnership with SHP2-GFP was followed in real-time bymeasuring the decrease in fluorescence emission at 520 nm. Reactionscontaining 0.5 μM SHP2-GFP, 50 mM Tris-HCl pH 7.2, 1 mM ATP, 10 mMMgCl₂, 10 mM μ-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij 35 and 40 μMpeptide were set up in a black microtitre plate. An initial equilibriummeasurement was made before adding 6 units of Src kinase, or buffer tocontrol wells. The decrease in fluorescence emission at 520 nm wasfollowed in real-time at 37° C. as shown in FIG. 20.

EXAMPLE 11 In Vivo Measurement of Kinase or Phosphatase Activity

The enzymatic activity of a kinase or phosphatase enzyme is measured inan in vivo assay performed as follows.

In vivo assays are carried out by transfecting cells with a firstexpression construct encoding a fusion protein comprising a polypeptidecomprising a natural binding domain and further comprising a site forphosphorylation fused in frame to a fluorescent protein and a secondexpression construct comprising a polypeptide comprising a bindingpartner for the natural binding domain fused in frame to a secondfluorescent protein. Alternatively, cells are transfected with a tandemconstruct encoding a fusion protein comprising a natural binding domainthat includes a site for phosphorylation and a binding partner for thenatural binding domain, and two different fluorescent proteins. For allexperiments, binding of a natural binding domain to its binding partneris dependent on phosphorylation or dephosphorylation.

Plasmids encoding the autofluorescent proteins (AFPs) red shifted greenfluorescent protein (rsGFP) and blue fluorescent protein (BFP) arepurchased from Quantum Biotechnologies, Inc. BFP is a mutated version ofthe 28 kDa rsGFP. BFP has an excitation peak of 387 nm and an emissionpeak of 450 nm. GFP has an excitation peak of 473 nm and an emissionpeak of 509 nm.

Constructs

DNA primers are designed encoding a first polypeptide comprising anatural binding and a phosphorylation site domain or a secondpolypeptide comprising a binding partner for the first polypeptide, astop codon and unique restriction sites (e.g. BamHI and EcoRI) at eachend to facilitate cloning. Codon usage is selected in order to allowboth mammalian and bacterial expression. Alternatively, DNA primers aredesigned to encode a polypeptide comprising both a natural bindingdomain that includes a phosphorylation site and a binding partner forthe natural binding domain.

Experiments are performed using the following pair of polypeptides:

1. v-SRC SH2 domain (amino acids 148-246; Waksman et al., supra; OWLdatabase accession no. M33292 and hamster polyomavirus middle T antigen(Ag) (321-331, EPQYEEIPIYL, SEQ ID NO: 17), Waksman et al., supra; OWLdatabase accession no. P03079,

2. Phosphotyrosine binding domain (PTB) of IRS-I (amino acids 157-267)Zhou et al., supra; OWL accession no. P35568 and interleukin 4 receptor(Il-4R) (anino acids 489-499, LVIAGNPAYRS, SEQ ID NO: 18; Zhou et al.,supra; OWL database accession no. P24394, and

3. The PTB domain of the proto-oncogene product Cbl (the Cbl N-termninalbinding domain) (amino acids 1-357); Lupher et al., supra; OWL accessionno. P22681 and a peptide derived from the Zap-70 tyrosine kinase (aminoacids 284-299, NH₃-IDTLNSDGYtpepARI- COOH, SEQ ID NO:19); Lupher et al.,supra; OWL accession no. P43403.

4. SH2 domain of ZAP70 (residues 1-259), GenBank accession No. L05148.Tandem phosphorylation motif of TCRseta chain (residues 52-163) GenBankaccession No. J04132.

Experiments are also performed using a construct encoding a polypeptidecomprising a natural binding domain including a site for phosphorylationand further comprising a natural binding partner for the natural bindingdomain.

These experiments are performed using the polypeptide c-SRC (residues86-536); Xu et al., supra; GenBank Accession No. K03218.

AEP-polypeptide construction

The purified DNA fragment, isolated by PCR is digested with theappropriate enzymes that cleave at the unique restriction sites locatedat each end and purified as above prior to ligation into the mammalianexpression vectors pQBI25-fcl and pQBI50-fcl.

The v-SRC-SH2 domain and the polyomavirus middle T-antigen peptide arecloned such that the AFP is placed at the N-termini. The AFPs can befused either to the N or C-termini of the PTB domain of IRS-1 and theIL-4R peptide.

In the case of the tandem construct expressing the c-SRC polypeptidewhich includes both a natural binding domain, including a site ofphosphorylation and a natural binding partner for the natural bindingdomain, chemical modification by the kinase or phosphatase enzyme beingassayed is monitored by measuring a change in an intramolecularreaction. A nucleic acid encoding a natural binding domain including asite of phosphorylation and its binding partner to be expressed as partof a single-polypeptide, additionally encodes, at a minimum, a donor AFPfused to the natural binding domain and an acceptor AFP fused to itsbinding partner, a linker that couples the two AFPs and is of sufficientlength and flexibility to allow for folding of the polypeptide andpairing of the natural binding domain, sequence or polypeptide with thebinding partner, and gene regulatory sequences operatively linked to thefusion coding sequence.

To prepare a construct encoding a polypeptide comprising a naturalbinding domain and further comprising a natural binding partner for thenatural binding domain and two different AFP proteins, the purified DNA(prepared as above) is ligated into a vector encoding an AFP. A fragmentencoding the polypeptide plus an AFP protein is excised from the vectorand ligated into a second vector encoding a linker and a second AFP.Alternatively, the purified DNA (prepare as above) is ligated into avector encoding an AFP. A DNA fragment encoding a linker and a secondAFP is ligated into the above construct (by digestion with appropriaterestriction enzymes) resulting in a construct encoding a polypeptidecomprising a natural binding domain, a natural binding partner for thenatural binding domain and two AFPs separated by a linker.

FRET in Mammalian cells

Experiments are performed using pairs of vectors expressing thefollowing proteins: a polypeptide comprising a natural binding domainincluding a phosphorylation site and an AFP and a polypeptide comprisinga natural binding partner of the natural binding domain and a secondAFP.

Vectors capable of expressing these proteins are transfected into COS-7cells (a well-established cell-line derived from monkey kidney cells)individually and in combination. Transfections are performed usingLipofectamine 2000 (GibcoBRL) and the transfected cells are incubated at37° C. for 48 hr (to allow the expressed proteins to accumulate to adetectable level) in the presence or absence of a kinase activator, acandidate modulator of kinase activity or both a kinase activator and acandidate modulator of kinase activity. Alternatively, the transfectedcells are incubated in the presence or absence of a phosphataseactivator, a candidate modulator of phosphatase activity or both aphosphatase activator and a candidate modulator of phosphatase activity.

Additional experiments are performed (using the transfection protocoldescribed above) using a tandem vector expressing a polypeptidecomprising a natural binding domain including a phosphorylation site, anatural binding partner of the natural binding domain and two differentAFPs separated by an appropriate linker. As above, transfected cells areincubated for 48 hrs in the presence or absence of a kinase, a candidatemodulator of kinase activity, both a kinase and a candidate modulator ofkinase activity, a phosphatase, a candidate modulator of phosphataseactivity or both a phosphatase and a candidate modulator of phosphataseactivity.

Following the 48 hr incubation period the amount of FRET is determinedby analysis in a BMG Galaxy fluorescent plate reader using the followingregime: excitation at 370 nm (excitation for BFP) and emission at 520 nm(emission for GFP). USE

The invention is useful in monitoring the activity of a protein kinaseor phosphatase, whether the protein is isolated, partially-purified,present in a crude preparation or present in a living cell. Theinvention is further useful in assaying a cell or cell extract for thepresence- or level of activity of a protein kinase or phosphatase. Theinvention is additionally useful in assaying the activity ofnaturally-occurring (mutant) or non-natural (engineered) isoforms ofknown protein kinases and/or phosphatases or, instead, that of novel(natural or non-natural) enzymes. The invention is of use in assayingthe efficacy of candidate modulators of the activity of a protein kinaseor phosphatase in inhibiting or enhancing the activity of that enzyme;moreover, is useful to screen potential therapeutic drugs for activityagainst cloned and/or purified enzymes that may have important clinicalpathogenicities when mutated. The invention is further of use in thescreening of a candidate bioactive agent (e.g., drugs) for side effects,whereby the ability of such an agent to modulate the activity of aprotein kinase or phosphatase may be indicative a propensity towardprovoking unintended side-effects to a therapeutic or other regimen inwhich that agent might be employed.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

20 1 12 PRT Homo sapiens 1 Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln SerVal 1 5 10 2 12 PRT Homo sapiens 2 Ala Val Gly Asn Pro Glu Tyr Leu AsnThr Val Gln 1 5 10 3 15 PRT Artificial Sequence Description ofArtificial Sequence Tumor suppressor protein p53 phosphorylation site 3Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys 1 5 10 15 411 PRT HAMSTER 4 Glu Pro Gln Tyr Glu Glu Ile Pro Ile Tyr Leu 1 5 10 5 11PRT Artificial Sequence Description of Artificial Sequence Interleukin 4receptor binding domain 5 Leu Val Ile Ala Gly Asn Pro Ala Tyr Arg Ser 15 10 6 16 PRT Artificial Sequence Description of Artificial SequenceZAP-70 binding domain 6 Ile Asp Thr Leu Asn Ser Asp Gly Tyr Thr Pro GluPro Ala Arg Ile 1 5 10 15 7 36 PRT Artificial Sequence Description ofArtificial Sequence Phosphorylated TCR-zeta chain 7 Arg Cys Lys Phe SerArg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln LeuTyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp35 8 33 DNA Artificial Sequence Description of Artificial SequenceForward PCR primer for SH2 domain of ZAP-70 8 gggatccata tgccagaccccgcggcgcac ctg 33 9 33 DNA Artificial Sequence Description of ArtificialSequence Reverse PCR primer for SH2 domain of ZAP-70 9 ggaattcgggcactgctgtt ggggcaggcc tcc 33 10 23 PRT Artificial Sequence Descriptionof Artificial Sequence Chk1 substrate peptide sequence 10 Lys Lys LysVal Ser Arg Ser Gly Leu Tyr Arg Ser Pro Ser Met Pro 1 5 10 15 Glu AsnLeu Asn Arg Pro Arg 20 11 31 PRT Artificial Sequence Description ofArtificial Sequence PKA peptide sequence 11 Glu Arg Glu Ile Lys Ala LeuGlu Arg Glu Ile Arg Arg Leu Arg Arg 1 5 10 15 Ala Ser Gln Ala Leu GluArg Glu Ile Ala Gln Leu Glu Arg Glu 20 25 30 12 32 PRT ArtificialSequence Description of Artificial Sequence PKA peptide sequence 12 LeuArg Gln Arg Ile Gln Cys Leu Arg Tyr Arg Ile Arg Arg Leu Arg 1 5 10 15Arg Ala Ser Gln Ala Leu Arg Gln Arg Ile Ala Gln Leu Lys Gln Arg 20 25 3013 36 PRT Artificial Sequence Description of Artificial SequencePhosphorylated TCR-zeta chain 13 Arg Cys Lys Phe Ser Arg Ser Ala Glu ProPro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu AsnLeu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp 35 14 36 PRTArtificial Sequence Description of Artificial Sequence Un-phosphorylatedTCR-zeta chain 14 Arg Cys Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala TyrGln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly ArgArg Glu Glu Tyr 20 25 30 Asp Val Leu Asp 35 15 40 PRT ArtificialSequence Description of Artificial Sequence SHPS-1 peptide sequence 15Lys Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn Leu Pro Lys 1 5 1015 Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn His Thr Glu 20 2530 Tyr Ala Ser Ile Gln Thr Ser Cys 35 40 16 40 DNA Artificial SequenceDescription of Artificial Sequence Forward PCR primer for SH2 domain ofSHP-2 16 ggggatcctc tagaatgaca tcgcggagat ggtttcaccc 40 17 33 DNAArtificial Sequence Description of Artificial Sequence Reverse PCRprimer for SH2 domain of SHP-2 17 ggggaattct ttcagcagca tttatacgag tcg33 18 11 PRT Artificial Sequence Description of Artificial Sequencev-SRC SH2 domain 18 Glu Pro Gln Tyr Glu Glu Ile Pro Ile Tyr Leu 1 5 1019 11 PRT Artificial Sequence Description of Artificial SequencePhosphotyrosine binding domain of IRS-1 19 Leu Val Ile Ala Gly Asn ProAla Tyr Arg Ser 1 5 10 20 9 PRT Artificial Sequence Description ofArtificial Sequence Phosphytyrosine binding domain of Cb1 20 Ile Asp ThrLeu Asn Ser Asp Gly Tyr 1 5

What is claimed is:
 1. A method of screening for a candidate modulatorof enzymatic activity of a kinase or a phosphatase, the methodcomprising a) contacting an isolated natural binding domain, a bindingpartner therefor and an enzyme with a candidate modulator of said kinaseor phosphatase, wherein said natural binding domain includes a site forpost-translational phosphorylation and binds said binding partner in amanner that is dependent upon phosphorylation or dephosphorylation ofsaid site by said kinase or phosphatase and wherein at least one of saidisolated natural binding domain and said binding partner comprises adetectable label; b) detecting the binding or dissociation of saidisolated natural binding domain to/from said binding partner; and c)monitoring the binding of said isolated natural binding domain to saidbinding partner, wherein binding or dissociation.of said isolatednatural binding domain and said binding partner as a result of saidcontacting is indicative of modulation of enzymatic activity by saidcandidate modulator of said kinase or phosphatase.
 2. The methodaccording to claim 1, wherein said detectable label emits light.
 3. Themethod according to claim 2, wherein said light is fluorescent.
 4. Themethod according to claim 1, wherein said monitoring comprises measuringa change in energy transfer between a detectable label present on saidisolated natural binding domain and a detectable label present on saidbinding partner.
 5. A method of screening for a candidate modulator ofenzymatic activity of a kinase or a phosphatase, the method comprisinga) contacting a cell-free assay system comprising an isolated naturalbinding domain and a binding partner therefor with a candidate modulatorof enzymatic activity of a said kinase or phosphatase; b) detecting thebinding or dissociation of said isolated natural binding domain to/fromsaid binding partner; and c) monitoring binding of said isolated naturalbinding domain and said binding partner therefor in said assay system,wherein said isolated natural binding domain includes a site forpost-translational phosphorylation and binds said binding partner in amanner that is dependent upon phosphorylation or dephosphorylation ofsaid site by a said kinase or phosphatase in said assay system, whereinat least one of said isolated natural binding domain and said bindingpartner comprises a detectable label, and wherein binding ordissociation of said isolated natural binding domain and said bindingpartner as a result of said contacting is indicative of modulation ofenzymatic activity by said candidate modulator of a said kinase orphosphatase.
 6. The method according claim 1 or 5, wherein said methodcomprises real-time observation of association of a said isolatednatural binding domain and its binding partner.